92 Physiolog3~ Biochemistry and Pharmacology

Reviews of 92 Physiolog3~ Biochemistry and Pharmacology

Editors R. H. Adrian, Cambridge. H. zur Hausen, Freiburg E. Helmreich, Wiirzburg • H. Holzer, Freiburg R. Jung, Freiburg • O. Krayer, Boston R. J. Linden, Leeds. P. A. Miescher, Gen~ve J. Piiper, G6ttingen • H. Rasmussen, New Haven U. Trendelenburg, Wiirzburg • K. Ullrich, Frankfurt/M. W. Vogt, G6ttingen • A. Weber, Philadelphia

With 13 Figures

Springer-Verlag Berlin Heidelberg New York 1982 ISBN 3-540-11105-0 Springer-Verlag Berlin Heidelberg New York ISBN 0-387-11105-0 Springer-Verlag New York Heidelberg Berlin

Library of Congress-Catalog-Card Number 74-3674 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustra- tions, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to 'Verwertungsgesellschaft Wort', Munich. @ by Springer-Verlag Berlin Heidelberg 1982 Printed in Germany. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant pro- tective laws and regulations and therefore free for general use. Offsetprinting and Binding: Konrad Triltsch, Wt~rzburg 2121/3130-543210 Contents

Cardioactive Substances that Prolong the Open State of Sodium Channels. By P. HONERJAGER, Mfinchen/Federal Re- public of Germany. With 12 Figures ....

Factors Influencing Renal Sodium Reabsorption in Volume Expansion. By F. G. KNOX and J. A. HAAS, Rochester, MN/USA ...... 75

Hormonal Coordination of the Immune Response. By J. COMSA, H. LEONHARDT, and H. WEKERLB, Homburg/Federal Republic of Germany. With 1 Figure .... : . . 115

Author Index ...... 193

Subject Index ...... 219

Indexed in Current Contents Rev. Physiol. Biochem. Pharmacol., Vol. 92 ©by Springer-Verlag 1982

Cardioactive Substances that Prolong the Open State of Sodium Channels

PETER HONERJAGER *

Contents

1 Introduction ...... 2

Chemistry of Na Channel-Gate Toxins ...... 4 2.1 Alkaloids ...... 4 2.2 Diterpenoids ...... 6 2.3 Polypeptides ...... 6

Mode of Action of Na Channel-Gate Toxins ...... 7 3.1 Methods of Analysis...... 7 3.2 Ceveratrum Alkaloids ...... 7 3.3 Batrachotoxin ...... 9 3.4 Aconitine ...... 10 3.5 Grayanotoxins ...... 10 3.6 Scorpion Toxins ...... 10 3.7 Sea Anemone Toxins ...... 11 3.8 Coral Toxin ...... 12 3.9 Silent Na Channels ...... 12 3.10 Antagonists ...... 13

4 Cardiac Action of Na Channel-Gate Toxins ...... 15 4.1 Role of Na Channels in the Cardiac Action Potential ...... 15 4.2 Ceveratrum Alkaloids ...... 19 4.3 Batrachotoxin ...... 37 4.4 Aconitine ...... 39 4.5 Grayanotoxins ...... 42 4.6 Scorpion Toxins ...... 44 4.7 Sea Anemone Toxins ...... 46 4.8 Coral Toxin ...... 50

Sarcolemmal Na Permeability and Myocardial Force of Contraction: Na-Ca Exchange ...... 50 5.1 Introduction ...... 50 5.2 Na-Ca Exchange in the Squid Giant Axon ...... 51 5.3 Nai-Ca o Exchange in Cardiac Preparations ...... 53

Comparison of Cardiac Actions of Na Channel-Gate Toxins with Those of Other Positive Inotropic Drugs ...... 56 6.1 Isometric Contraction Curve ...... 57 6.2 Blockade of Sarcolemmal Na Channels by Tetrodotoxin ...... 58

* lnstitut fiir Pharmakologie und Toxikologie der Technischen Universit~it Mfinchen, Biedersteiner StratSe 29, D-8000 Mtinchen 40, Federal Republic of Germany 2 P. Honerj~iger

6.3 Contraction Frequency ...... 60 6.4 Reduction of Extracellular Na Concentration ...... 60 6.5 Variation of Extracellular K Concentration ...... 61 6.6 Inhibition of the Na Pump by Dihydro-Ouabain ...... 61 6.7 Propranolol ...... 62

7 Summary and Conclusions ...... 62

References ...... 64

Abbreviations and Symbols

ATXII Toxin II fromAnemoniasulcata h Inactivation variable of Iqa cAMP Cyclic adenosine 3',5'-mono- channels phosphate m Activation variable of Na D 600 o~-Isopropyt-c~-[(N-met hyl-N- channels homoveratryl)-'),-aminopropyl]- I K Transmembrane/( current 3,4,5-trimethoxyphenylaceto- INa Transmembrane Na current nitrile (gallopamil) through Na channels ScTXII Toxin II from Androctonus Isi Transmembrane slow inward australis Hector (Ca, Na) current STX Saxitoxin PNa Membrane permeability to Na TTX Tetrodotoxin ions *max Maximal rate of depolarization during the upstroke of the F c Peak force of contraction action potential AF c Change in peak force of con- [ ]a Extraeellular concentration traction [ 1; Intracellular concentration

1 Introduction

It is now recognized that many toxic substances evolved by plants or animals act by interfering with the excitation process of nerve and muscle cells. Specifically, these compounds alter the rapid and transient increase in membrane permeability to Na ions (PNa), that initiates the action potential. According to the general mode by which toxins modify the Na permeability mechanism, or Na channels, two groups can be distinguished. The first group of substances, including tetrodotoxin (TTX) and saxitoxin (STX), blocks the flow of Na ions through Na channels. The second group of toxins maintains an open form of the Na channel, thereby prolonging the time during which Na ions flow into the cell. This group com- prises alkaloids (ceveratrum alkaloids, batrachotoxin, aconitine), the diterpenoid grayanotoxins, and polypeptides from scorpions, sea anemones and coral and contains the most potent and most specific substances known to increase the Na permeability of excitable membranes. Toxins Cardioactive Substances that Prolong the Open State of Sodium Channels 3 which prolong the open state of Na channels will be referred to as Na channel-gate toxins to denote the specific function of the Na channel affected by these agents, i.e. the gating mechanism which controls the activation and inactivation of Na channels. While the general pharmacology of one subgroup of Na channel-gate toxins, the ceveratrum alkaloids, has been studied for more than a century, the basic mechanism of their action, as outlined above, has been elucidated only during the last 2 decades following the discovery of the ionic permeability changes responsible for membrane excitation (Hodgkin and Huxley 1952). For methodological reasons we possess more detailed information concerning the effect of Na channel-gate toxins on nerve fibres and nerve cells in culture than on myocardial and other excitable cells. This review, therefore, includes a brief description of effects on the type of preparation which yielded most information on the mechanism of action at the level of Na channels. For in-depth information of the effects of toxins on Na channels, the reader is referred to the reviews by Ulbricht (1969, 1974), Hille (1970), Narahashi (1974), Khodorov (1979), Ritchie (1979), Catterall (1980), and Lazdunski et al. (1980), and to the original literature cited in connection with individual toxins. This review deals with the direct cardiac effects of Na channel-gate toxins observed in isolated cardiac preparations. The main effects are a positive inotropic effect without a significant chronotropic effect and an arrhythmogenic effect. Early references to the direct cardiac effects of ceveratrum alkaloids are given in the reviews by Krayer and Acheson (1946) and Trautwein (1963). Trautwein (1963) also reviewed the literature concerning the cardiac effects of aconitine. The Benforado review (1967) includes a chapter on the direct cardiac effects of ceveratrum alkaloids and concludes that "the mechanism by which the veratrum alkaloids exert their positive inotropic action on the heart is not known". The last 15 years have brought considerable progress in our understanding of the basic membrane action of ceveratrum alkaloids and other Na channel-gate toxins and of the relationship between ion fluxes through the sarcolemma and myocardial force of contraction. Perhaps the most impressive result is that the large group of chemically diverse Na channel- gate toxins represents a single class of inotropic agents. They possess a characteristic mode of action that differs from that of other cardioactive drugs, e.g., cardioactive steroids, catecholamines, and other agents thought to act by influencing the cellular concentration of cyclic nucleotides. In the intact animal organism, the direct cardiac effects of Na channel- gate toxins may not be recognized because of cardiovascular or cardio- cardiac reflexes following the stimulation of sensory nerves by the toxins. The Bezold-Jarisch effect exemplifies this action for many ceveratrum 4 P. Honerjfiger alkaloids (Krayer 1961). The hypotensive and other circulatory effects of ceveratrum alkaloids were reviewed by Benforado (1967), Kupchan and Flacke (1967), and Krayer and Meilman (1977). The older literature on this subject was surveyed by Krayer and Acheson (1946) in their com- prehensive review of the pharmacology of veratrum alkaloids. Notably, certain Na channel-gate toxins lack a hypotensive effect; for example, some natural and semi-synthetic ceveratrum alkaloids (Kupchan and Flacke 1967) and at least one of the polypeptide toxins, namely anthopleurin A, which has a direct positive inotropic effect on the myocardium in conscious dogs (Blair et al. 1978).

2 Chemistry of Na Channel-Gate Toxins

2.1 Alkaloids

2.1.1 Ceveratrurn Alkaloids The veratrum alkaloids are steroid alkaloids that occur in liliaceous plants belonging to the genera Veratrum, Zygadenus, Stenanthiurn, and Schoenocaulon. These genera comprise a section of the suborder Melan- thioideae (Kupchan et al. 1961). The terms ceveratrurn and jerveratrurn alkaloids were introduced to distinguish two chemically and pharmacologically different groups of veratrum alkaloids (Fieser and Fieser i 959). The jerveratrum alkamines are secondary amines, contain only one to three atoms of oxygen, and are found in unhydrolyzed plant extracts in part as the free alkamines and in part in combination with one molecule of D-glucose as glucoalkaloids. Jerveratrum alkaloids do not prolong the open state of Na channels. Somej erveratrum alkaloids, such as veratramine, antagonize the effect of ceveratrum alkaloids (Krayer and George 1951), probably because they block Na channels (Ohta et al. 1973). The ceveratrum alkamines are tertiary amines, are highly hydroxylic, and contain seven to nine atoms of oxygen. They usually occur esterified with various acids as ester alkaloids, but are sometimes unconjugated. They have never been found as glycosides. The ceveratrum alkaloids can be formally derived from (22 S, 25 S)-5/3-cevanine. Their chemistry has been reviewed by Kupchan and By (1968). The structures of the monoester veratridine and the triester germitrine are shown in Fig. 1. The name veratrine is usually used for the total alkaloids of the seeds of Schoenocaulon offici- nale, it does not refer to a well-defined preparation of constant composition (Krayer and Acheson 1946; Kupchan et al. 1961). The use of this and other mixtures of veratrum alkaloids for scientific purposes is dis- couraged. Jerveratrum alkaloids may antagonize the effect of ceveratrum Cardioactive Substances that Prolong the Open State of Sodium Channels 5

Fig. 1. Chemical structures of CH3H the ceveratrum alkaloids veratridine and germitrine. (Drawn after Kupchan and By 1968) N?H/N~IH3

OCHa / ~/;"-HI~/ "OH

OH^O-(/ ')-CO" ~ J ~/

Veratridine CH3H

N?/N~cH 3

0 r/ "4/ ~ "~/ i"/ "OH .L -°l°"i o,

CH3 0 0 CH3

Germitrine

alkaloids, and there are more subtle differences between the effects of individual ceveratrum alkaloids (veratridine versus cevadine; Reiter 1963; veracevine and germine and their esters, Sect. 3.2; veratridine versus germitrine, Sect. 4.2.5).

2.1.2 Batrachotoxin Batrachotoxin is the most toxic of the steroid alkaloids extracted from the skin of the Columbian arrow poison frog (Phyllobates aurotaenia). It is a pyrrolecarboxylate ester of the pregnane derivative batrachotoxi- nin A (Tokuyama et al. 1969).

2.1.3 Aconitine Aconitine is the main alkaloid of Aconitum napellus of the plant family Ranunculaceae (List and HOrhamrner 1969). Like the ceveratrum alkaloids, aconitine is an ester of an alkamine with organic acids. The alkamine, 6 P. Honerj~iger aconine, contains two cyclopentane rings, one piperidine ring, and three cyclohexane rings. In aconitine, aconine is esterified with benzoic acid and acetic acid. The structure of aconitine was reported by Wiesner et al. (1969).

2.2 Diterpenoids

Gyanotoxins are the toxic principles found in the leaves of various species of Rhododendron, Kalmia, and Leucothoe (Ericaceae). They are diterpenoids with a perhydroazulene skeleton and their chemical structures have been determined (Kakisawa et al. 1965; Kumazawa and Iriye 1970).

2.3 Polypeptides

2. 3.1 Scorpion Toxins Toxins of North African scorpions were first purified, characterized, and sequenced by Rochat, Miranda, Lissitzky, and colleagues (Miranda et al. 1970; Rochat et al. 1970; Kopeyan et al. 1979). These toxins are basic polypeptides with molecular weights of approximately 7000. Each scorpion species studied contained multiple toxins having extensive sequence homology (Rochat et al. 1970). The molecular weight of toxin II from Androctonus australis HECTOR (ScTXII) is 7249 (Miranda et al. 1970).

2.3.2 Sea Anemone Toxins Anemone toxins were first isolated in pure form from Anemonia sulcata, shown to be basic polypeptides of 2500-5000 molecular weight and sequenced by Bdress, Wunderer, and their colleagues (BOress et al. 1975a,b, 1977; Wunderer et al. 1976). The sequences of three toxins from Ane- monia sulcata and one from Anthopleura xanthogrammica were determined (Wunderer et al. 1976; B~ress et al. 1977; Martinez et al. 1977; Wunderer and Eulitz 1978; Tanaka et al. 1977). Toxins I and II from Anemonia sulcata and anthopleurin A fromAnthopleura xanthogrammica consist of 47-49 amino acid residues and have substantial sequence homology. None of the anemone toxins has detectable sequence homology when compared to the scorpion toxins.

2. 3. 3 Coral Toxin Goniopora toxin, a polypeptide with a molecular weight of 12 000, was isolated and purified from the coral Goniopora spp. (Hashimoto and Ashida 1973). Cardioactive Substances that Prolong the Open State of Sodium Channels 7

3 Mode of Action of Na Channel-Gate Toxins

3.1 Methods of Analysis

Different methods have been employed to study the effects of drugs on Na channels; electrophysiologic analysis of membrane voltage and membrane current, isotopic flux measurements of ion permeability, and direct binding studies of radiolabelled toxins. The results obtained with the latter two methods were recently reviewed (Catterall 1980), and have helped significantly to elucidate molecular aspects of the effects of toxins on the Na channel. The electrophysiologic approach, with its advantage of high time resolution, provides information allowing us to predict how the Na channel-gate toxins modify the transmembrane electrical activity of cells. This is particularly important for an understanding of the cardiac effect of these agents. The effects on the kinetics of Na channels are therefore reviewed in some detail. It has become customary to analyse the effects of drugs on Na channels in terms of the conceptu- alization introduced by Hodgkin and Huxley (1952) to describe the normal Na permeability changes in the squid giant axon. According to this theory, two voltage- and time-variant parameters, the activation variable rn and the inactivation variable h, govern the changes of the Na permeability during excitation. The Na permeability is turned on because of an increase of rn if the membrane is depolarized, and the turning off during maintained depolarization is due to a decrease of h. Repolarization turns off PNa because of a decrease in rn. This analysis was also used with some modifications to describe the behaviour of Na channels in other types of excitable cells, including cardiac cells (see Trautwein 1973). The hypothetical subunit of the Na channel, which gives adequate kinetics of opening and closing, is referred to as the gating mechanism, consisting of the rn gate and the h gate.

3.2 Ceveratrum Alkaloids

The veracevine ester veratridine has received the most attention among the various ceveratrum alkaloids. A detailed analysis of the effect of veratridine on the frog node of Ranvier was performed by Ulbricht (1969, 1972a,b) using the voltage-clamp technique. Normally, the Na permeability of nerve fibres exhibits a fast and transient rise of about 1 ms duration during a voltage-clamp depolarization. In contrast, the PNa observed in the veratridine-treated node, in addition to the normal Na permeability, develops over a period of several seconds during depolarization and is maintained as long as the membrane is clamped at the depolarized level. 8 P. Honerj~iger

The veratridine-induced PNa was quantitatively described by introducing an activation variable s (analogous to m) exhibiting the potential- and time-dependence observed and by omitting an inactivation term corresponding to h (UIbricht 1969). For a stepwise depolarization, the time course of s follows the equation s(t ) = s(t = ~o) (1 - e- t/rs), where t is the time after the start of the depolarization and r s is a time constant. The time constant of activation of the veratridine-induced PNa (~-s) is of the order of seconds, i.e. about 104 -times larger than rm, the time constant of the normal PNa activation. The voltage-dependence of the veratridine- induced PNa activation is less steep and shifted to more negative membrane potentials compared to that of normal PNa" Veratridine seems to modify some of those Na channels participating normally in the production of the action potential rather than to create new channels (Ulbricht 1969). This would explain why the appearance of the alkaloid-induced PNa is accompanied by a decrease in the normal transient PNa" Furthermore, normal and modified channels distinguish to comparable degrees between Na, Li, choline, and tromethamine, and both are blocked by TTX at rates and to extents that are very similar (Ulbricht 1974). The concentration of veratridine appears to determine the proportion of Na channels that is modified, while at the level of the single Na channel the interaction with veratridine appears to be a reversible all-or-none event (Ulbricht 1972a). At concentrations exceeding about 10 pmol/litre veratridine induces a sustained depolarization of the node of Ranvier due to an increase in PNa (Ulbricht 1969). It is caused by the two effects of veratridine on Na channels; block of inactivation and shift of activation to more negative potentials, thus allowing the spontaneous opening of Na channels at the level of the normal resting potential. Some pharmacologically important effects of veratridine occur at lower concentrations when the resting membrane potential shows very little, if any depolarization. At this stage of veratridine action, the presence of modified Na channels is apparent as a distinct delay in the final phase of repolarization of the action potential (after-depolarization). The effect reflects the large value of r s that determines the rate at which modified Na channels deactivate after repolarization (Ulbricht 1969). By this mechanism, veratridine greatly prolongs the phase of increased PNa and Na influx associated with each action potential. Other ceveratrum alkaloids are similar to veratridine in producing after- depolarizations in nerve fibres, but individual alkaloids differ with respect to duration (Graham and Gasser 1931; Shanes 1952;Honerfiiger 1973). The alkamines germine and veracevine induce after-depolarizations of relatively short duration in crustacean nerve axons. The half-life of after-depolarization decay is 6 8 ms (Honer]alger 1973). In the same Cardioactive Substances that Prolong the Open State of Sodium Channels 9 study it was shown that number and nature of the ester groups attached to the alkamine modify the duration of after-depolarization. For example, replacement of the 3~-hydroxyl in veracevine by an acetyl (cevacine), angeloyl (cevadine), or veratroyl group (veratridine) causes a progressive prolongation of the after-depolarization up to about 3 s (half-life) in the case ofveratridine. Even longer-lasting after-depolarizations were observed with the di-, tri-, and tetraester alkaloids in guinea-pig papillary muscle (Sect. 4.2.5). The semisynthetic germine ester germine-3-acetate produces an after-depolarization in curstacean nerve fibres that decays with a half- life of 40-70 ms. Voltage-clamp experiments on squid axons showed that this alkaloid induces a TTX-sensitive slow component of Na permeability (Honer]alger 1973). After repolarization this component of PNa deactivates about 20-times faster than the PNa induced by veratridine. This may indicate that all the various ceveratrum alkaloids react with Na channels, but differ in the degree to which they slow the m-gating process.

3.3 Batrachotoxin

A detailed voltage-clamp analysis carried out on the frog node of Ranvier showed that batrachotoxin alters both the gating mechanism and ion selectivity of Na channels (Khodorov and Revenko 1979). The Na channels lose the ability to inactivate, and the voltage-dependence of activation is shifted by about 50 mV to more negative voltages. Therefore, a fraction of Na channels is permanently activated at the normal level of resting potential, resulting in depolarization. The time constant of activation is also affected by batrachotoxin, but to a much lesser extent than by veratridine. Batrachotoxin increases the relative permeability of the Na channels toward larger ions, including Ca ions. This finding, by Kho- dorov and Revenko (1979), is of particular interest with regard to the positive inotropic effect on myocardial cells of batrachotoxin, as well as other Na channel-gate toxins (Chap. 4). If myocardial Na channels were to lose their ionic selectivity and become significantly permeable to Ca ions, this would explain a positive inotropic effect of the Na channel-gate toxins. It seems, however, that this effect of batrachotoxin on neural Na channels cannot be extended to cardiac Na channels, at least as far as the action of veratridine and ScTXII is concerned. The Ca influx induced by these Na channel-gate toxins in cardiac cells in culture does not occur through Na channels, but by way of Na-Ca exchange (Sect. 5.3.2). Khodorov and Revenko (1979) further showed that the modification of Na channels by batrachotoxin requires a specific functional state of the Na channels, namely their open configuration. Thus the effect develops 10 P. Honerj~iger much more rapidly if the node of Ranvier is stimulated repetitively in such a way that the Na channels are not inactivated by the holding potential and the pulses are large enough to activate Na channels. The concen- tr~ition-effect relationship suggests that each channel receptor binds only one molecule of batrachotoxin. A study of the effect of batrachotoxin on the action potential of guinea-pig papillary muscle led to the same conclusion (Sect. 4.3.2).

3.4 Aconitine

The mode of action of aconitine on Na channels was elucidated in voltage- clamp experiments on the frog node of Ranvier (Schmidt and Schmitt 1974; Mozhayeva et al. 1977), and was found to be very similar to that of batrachotoxin. Aconitine-modified Na channels lose the property of inactivation and open at more negative potentials than the normal resting potential. These channels open with a time constant about twice as large as normal Na channels. As with batrachotoxin, the development of the effect is speeded up by periodical depolarizations if they lead to an activation of Na channels. This suggests that the open configuration of the Na channel gate is more favourable for the interaction with aconitine. Aconitine, like batrachotoxin, reduces the selectivity of Na channels for monovalent cations (Mozhayeva et al. 1977).

3.5 Grayanotoxins

Grayanotoxins have been found to depolarize the membranes of various excitable cells (see Starkus and Narahashi 1978). As shown on the squid giant axon, this is due to a selective and TTX-sensitive increase in resting PNa (Narahashi and Seyama 1974). The effect of grayanotoxins on the time- and voltage-dependence of activation and inactivation of Na channels has not been published in detail.

3.6 Scorpion Toxins

Venom of Leiurus quinquestriatus delays the repolarization of the action potential, reduces the resting membrane potential and induces spontaneous activity in myelinated nerve fibres (Adam et al. 1966). Voltage-clamp experiments on the frog node of Ranvier showed that the main effect of the venom is to slow the inactivation of Na channels (KoppenhOfer and Schmidt 1968a,b). Venoms or toxins of other North African scorpions Cardioactive Substances that Prolong the Open State of Sodium Channels 11

(Buthus thamulus, B. eupeus, Androctonus australis HECTOR) have the same principal effect on nerve fibres (see Khodorov 1979). In the squid giant axon, which allows application of substances to either side of the axolemma, venom of B. thamulus is effective only if applied from the extracellular side (Narahashi et al. 1972). More recently, Catterall (1980) showed that a toxin purified from L. quinquestriatus venom increases the fraction of Na channels activated by veratridine, aconitine, or grayanotoxin I in cultured neuroblastoma cells: His findings indicate that scorpion toxin and the group of alkaloidal and diterpenoid toxins (lipid- soluble toxins) bind to separate receptor sites of the Na channel which interact allosterically. In contrast to the venoms of North African scorpions, which modify Na inactivation, venom of the American scorpion Centruroides sculpturatus alters the activation gate without modifying the h gate in the frog node of Ranvier (Cahalan 1975). This venom causes a shift of the activation voltage-dependence by 40-50 mV in the hyperpolarizing direction, while the modification of the Na channel requires depolarization. While many Na channel-gate toxins alter the function of isolated cardiac muscle by modifying sarcolemmal Na channels, scorpion toxins seem to act mainly by releasing neurotransmitters from intracardiac nerve fibres, i.e. by their effect on neural Na channels (Sect. 4.6).

3.7 Sea Anemone Toxins

3. 7.1 Toxin H from A nemonia sulcata (A TXII)

In the crayfish giant axon, ATXII has little or no effect on resting potential, but produces a marked prolongation of the falling phase of action potentials (Rathmayer and B~ress 1976). As shown in the frog node of Ranvier, and crayfish giant axons, the main effect of ATXII is to slow the rate of the Na channel inactivation (Bergman et al. 1976; Romey et al. 1976). The activation of Na channels is not markedly altered. The curve relating steady-state inactivation to membrane potential shows a decrease in slope under the influence of ATXII, so that moderate depolarizations inactivate a larger than normal fraction of Na channels. The receptor for ATXII does not seem to be accessible from inside the axolemma. Like scorpion toxin, ATXII increases the fraction of Na channels modified by veratridine (Catterall 1980). Scorpion toxin and ATX n appear to act at a common receptor site associated with the Na channel (Catterall 1980). 12 P. Honerj~iger

3. Z2 Anthopleurin A Low concentrations of anthopleurin A produce slow transient depolarizations and a prolongation of the repolarization phase in the crayfish giant axon, while high concentrations induce a sustained depolarization (Low et al. 1979). Voltage-clamp experiments show that anthopleurin A alters the kinetics of Na channels (Low et al. 1979). Modified Na channels are activated at more negative membrane potentials, and the inactivation is slowed as is the removal of activation after repolarization.

3.8 Coral Toxin

Goniopora toxin prolongs the falling phase of the action potential in rabbit myocardium (Fu]iwara et al. 1979), and this effect is abolished by TTX. This is so far the only available evidence indicating that the toxin slows the kinetics of Na channels.

3.9 Silent Na Channels

The basic effect of the various Na channel-gate toxins was discovered in excitable membranes that produce their action potential by the activation of Na channels. However, it is becoming increasingly evident that neither the presence of a Na channel-dependent mechanism of excitation, nor excitability as such are prerequisites for Na channel-gate toxins to cause the appearance of persistently activated Na channels. Veratridine opens Na channels in the pancreatic I3-cell membrane which normally produces impulses by the activation of Ca channels (Donatsch et al. 1977). The action potential of chick embryonic heart cells in an early stage of development is due to the activation of TTX-insensitive, slow Na-Ca channels. Nevertheless, veratridine depolarizes these cells and the effect is blocked by TTX (Sperelakis and Pappano 1969; Bernard and Couraud 1979; Romey et al. 1980). Polypeptide toxins also produce bioelectric effects that are blocked by TTX in slow cells of the chick embryo heart (Bernard and Couraud 1979; Romey et al. 1980). These effects are described in detail in Chap. 4. Veratridine produces a TTX-sensitive depolarization in non-spiking sensory dendrites of the crab (Lowe et al. 1978). In an inexcitable cell line derived from a rat brain tumor, veratridine or polypeptide toxins open TTX-sensitive Na channels (Romey et al. 1979). When exposed to Na channel-gate toxins, fibroblasts exhibit an increased uptake of Na that is blocked by TTX (Munson et al. 1979;PouyssOgur et al. 1980). There are conflicting reports as to whether Na channel-gate toxins open Na channels in glial cells (Villegas et al. 1976; Tang et al. 1979). Cardioactive Substances that Prolong the Open State of Sodium Channels 13

As it is highly unlikely that the Na channel-gate toxins create Na-selective and TTX-sensitive ionic channels in these membranes, it is generally assumed that Na channels pre-exist in a state which under normal conditions precludes their electrical activation. The term silent Na channels has been suggested by Romey et al. (1979). If these Na channels were in a state characterized by faster inactivation kinetics than activation kinetics, this would explain why they are normally electrically silent, but able to open in the presence of a toxin that specifically slows the inactivation (Romey et al. 1979).

3.10 Antagonists

3.10.1 Non-Competitive Antagonists TTX and STX are potent and selective blockers of Na channels and they have no other known action (Narahashi 1974). TTX and STX also block the flow of Na ions through Na channels modified by Na channel-gate toxins, and thus are specific antagonists of Na channel-gate toxins. The blockers and the gate toxins modify different properties of the Na channel. TTX and STX, in partially blocking concentrations, reduce the total available Na permeability, but do not alter the kinetics of the unblocked Na channels. In contrast, the gate toxins alter the kinetic (m and/or h) behaviour of the Na channels. This difference suggests different receptor sites at the Na channel for blockers and gate toxins respectively, and thus a non-competitive type of antagonism. Catterall (1975) investigated the nature of this antagonism by using the initial rate of Na influx into cultured neuroblastoma cells to quantify the effect of veratridine or batrachotoxin. He has shown that activation of Na influx by the Na channel- gate toxins is non-competitively inhibited by TTX. Further proof that the receptors for TTX and Na channel-gate toxins are not identical was obtained in experiments on the specific binding of labelled TTX to nerve membrane preparations. This binding (and that of labelled STX) was unaffected even by high concentrations of Na channel-gate toxins (see Catterall 1980). Although the TTX-sensitivity of biological effects points to the involvement of Na channels, there are some instances in which the lack of sensitivity to TTX does not rule out the participation of Na channels. Examples of cells possessing TTX-insensitive Na channels are denervated skeletal muscle fibres (Redfern et al. 1970) and giant neurons of the snail Helix aspersa (Chamberlain and Kerkut 1969). These TTX-insensitive Na channels are susceptible to the action of Na channel-gate toxins such as veratridine (Leicht et al. 1971) or batrachotoxin (Albuquerque and Warnick 1972). 14 P. Honerj~iger

Normal Na channels and those modified by veratrine are blocked by the local anaesthetic lidocaine (frog node of Ranvier; Hille 1968). Lido- caine and tetracaine inhibit veratridine-induced Na uptake by embryonic cardiac cells (Fosset et al. 1977). An increase in external Ca concentration is known to reduce the depolarizing effect of veratridine (Ulbricht 1969). To understand this antagonism it is necessary to recall the effect of external Ca ions on normal Na channels. An increase in [Ca]o shifts the voltage dependence of both activation and inactivation to less negative membrane potentials (Frankenhaeuser and Hodgkin 1957). Varatridine-modified Na channels lose the inactivation gate, but their activation gate is similarly sensitive to changes of [Ca]o as that of normal Na channels (frog node of Ranvier; Ulbricht 1969). Thus, increasing [Ca]o at a given level of depolarization reduces the fraction of activated veratridine-modified Na channels and may lead to repolarization. This may also explain why concentrations of external Ca higher than 2 mmol/litre inhibit the veratridine-induced Na uptake by embryonic cardiac cells (Fosset et al. 1977). It is not necessary to assume a specific, e.g. competitive, interaction between Ca ions and veratridine to account for this antagonism. However, the inhibition by external Ca ions of the veratridine-induced Na influx into neuroblastoma cells (Catterall 1975) and cardiac cells in culture (Galper and Catterall 1979) appears to be of the competitive type.

3.10.2 Competitive Antagonists

The clarification of a pharmacological antagonism usually requires estab- lishing complete concentration-effect relationships in systems where the effect is a more or less direct measure of the degree of receptor occupancy by the drug. In the case of Na channel-gate toxins, Catterall (1975)was the first to obtain complete concentration-effect relationships for their effect on Na permeability by using the stimulation of 22 Na influx in cultured neuroblastoma cells. With this system he showed that there are competitive interactions among veratridine, batrachotoxin, aconitine, and grayanotoxin I (see Catterall 1980). The effect of saturating concentrations of batrachotoxin is reduced by each of the other lipid-soluble toxins, suggesting that batrachotoxin is a full agonist, whereas veratridine, aconitine, and grayanotoxin are partial agonists acting at a common receptor site of the Na channel. A competitive antagonist without notice- able intrinsic effect on the Na channels has apparently not yet been found. Cardioactive Substances that Prolong the Open State of Sodium Channels 15

4 Cardiac Action of Na Channel-Gate Toxins

4.1 Role of Na Channels in the Cardiac Action Potential

4.1.1 Na Channels in Different Cell Types of the Heart

During the last 15 years the application of voltage-clamp techniques to cardiac tisssues has provided much information on the transmembrane ionic currents underlying the action potential of cardiac cells, which is important for an understanding of the cardiac action of Na channel-gate toxins. The reader is referred to the reviews by Trautwein (1973) and Reuter (1979) and to the articles by McAllister et al. (1975) and by Beeler and Reuter (1977). In the case of mammalian ventricular myocardium, the action potential has been shown to result from the sequential and transient activation of a fast inward Na current (INa), a secondary, slow inward current carried by Ca and Na ions, (Isi), and an outward K current (IK) (Trautwein 1973; Reuter and SchoIz 1977; McDonald and Trautwein 1978). The fast PNa system responsible for INa is very similar to that of nerve cells and the term Na channels, originally applied to the ionic pathwahs of the Na conductance mechanism of nerve cells (Hille 1970), is now also used for that of cardiac cells (Hondeghem and Katzung 1977). The rapid upstroke of the action potential is caused by the rapid increase of INa , and the first time derivative of membrane potential during the upstroke (V) provides an indirect estimate of INa (Reuter 1979; Walton and Fozzard 1979). The electrotonic current resulting from the potential change during the upstroke is the stimulus for excitation of adjacent cells. Therefore, INa is the basis for conduction of the action potential, and conduction velocity is largely determined by the Vmax of the action potential. INa subsides completely within a few milliseconds after its activation, but the membrane potential remains at a positive level because of the now-apparent activation of Isi. The subsequent repolarization process is determined by the inactivation of Isi and the activation of I K . As in ventricular myocardial cells, a fast, inward Na current is responsible for the upstroke of the action potential in Purkinje fibres and atrial muscle fibres (Trautwein 1973). The action potential of Purkinje fibres exhibits a higher value of Vmax than the fibres of the working myocardium. This allows the Purkinje fibre to play its physiological role in dis- tributing excitation, leading to the nearly simultaneous contraction of the different parts of the ventricles. In Purkinje fibres, INa is not completely inactivated after the upstroke and participates in the ionic currents flowing during the plateau phase (Attwell et al. 1979). Thus, the action potential duration of Purkinje fibres, but not of ventricular muscle fibres, is markedly shortened by the specific Na channel blocker TTX 16 P. Honerj~iger

(Coraboeuf et al. 1979). The slowly rising action potentials of cells of the sinoatrial node are insensitive to TTX (Yamagishi and Sano 1966) indicating that Na channels are not involved in producing the rising phase of the action potential. Yet the presence of Na channels in this cell type is indicated by the observation that hyperpolarization of pacemaker cells by application of carbachol produces an increase in Vmax of the action potential that is entirely suppressed by TTX (Kreitner 1975). Hyperpolar- ization by the voltage-clamp technique also reveals a fast Na current that is inactivated by depolarization and blocked by TTX (Noma et al. 1977). The upstroke of the action potential of atrioventricular nodal fibres shows two components (Ruiz-Ceretti and Ponce Zurnino 1976). The first of these is attributed to the activation of Na channels because it depends strongly on [Na]o and is inhibited by TTX. Its relatively low Vmax of about 13 V/s is accounted for by the low resting potential of nodal cells causing inactivation of the Na channels. The voltage-dependence of Vrnax of atrioventricular nodal action potentials corresponds to that of Na channels (Shigeto and Irisawa 1974). The second component of the action-potential upstroke is attributed to the slow inward current (Ruiz- Ceretti and Ponce Zumino 1976). In summary, electrophysiologic experiments revealed the presence of Na channels in the plasmalemma of cells of the sinoatrial and atrioventricular nodes, atrial and ventricular myocardium, and of Purkinje fibres. Na channel gate toxins are expected to modify the electrical activity of all cells possessing Na channels unless the Na channels lack the pharma- cologic receptor for alteration of the gating mechanism by the toxin. The effects of Na channel-gate toxins on membrane currents of cardiac cells under voltage-clamp conditions are largely unknown. However, there is substantial indirect evidence obtained by intracellular potential recording suggesting that these toxins affect the kinetics of INa in a way similar to their effect on neural Na channels. A slowing of INa inactivation, as produced in the nerve membrane by sea anemone or scorpion toxins, or the removal of inactivation combined with a slowing of activation in a fraction of Na channels, as produced by veratridine in the node of Ranvier, would add a component of Na current to the membrane currents flowing during the plateau and repolarization phases of the cardiac action potential. This should drive the membrane potential from the normal level determined by the balance of Isi and I K towards the equilib- rium potential for Na ions (approx. + 50 mV) until the delayed inactivation of the modified Na channels and normal repolarizing mechanisms allow a return of the membrane potential to the normal resting level. Indeed, a prolongation of the repolarization phase is the characteristic effect on cardiac cells of many Na channel-gate toxins. Strong evidence for a selective modification of INa by these agents is the prevention of Cardioactive Substances that Prolong the Open State of Sodium Channels 17 their effect on the action potential in Na-poor medium or in the presence of TTX, i.e. under conditions where INa is absent and the action potential occurs through the activation of Isi.

4.1.2 Delayed Repolarization and Oscillatory After-Potentials The repolarization of the cardiac action potential proceeds in certain phases that are affected differently by individual Na channel-gate toxins. The first or early phase of repolarization to be distinguished in Purkinje fibres occurs immediately after the rapid upstroke and extends from the spike to the beginning of the plateau phase. This early repolarization, which is absent or much less pronounced in atrial and ventricular muscle fibres, has received scant attention in the study of Na channel-gate toxins and seems to be little affected by these agents (Schmidt 1960; Heistracher and Pillat 1962; Hogan and Albuquerque 1971 ; Ar bel et al. 1975; Shimizu et al. 1979). The early phase of repolarization is followed by the plateau phase and a phase of rapid repolarization during which the resting potential level is approached. In purely descriptive terms, there are two ways in which the course of repolarization following the plateau phase may be prolonged, and Na channel-gate toxins can be classified on this basis. First, repolarization can be slowed by a prolongation of the plateau phase, i.e., by a broadening of the action potential at nearly all potential levels between the peak of the plateau potential and the resting potential. Second, repolarization can be slowed, more or less abruptly, at any point during the fast phase of repolarization which follows the plateau phase; the latter and a variable, initial part of the former then remain unaffected. This second modification is usually called depolarizing after-potential or after-depolarization. These terms should replace the term negative after- potential (e.g., Fig. 1 in Shanes 1958), which was introduced when electrical activity was measured with extracellular electrodes. The intracellular potential is changed in the positive direction during an after-depolarization. It should be noted that a slowing of the rate of repolarization may have either an anti-arrhytl~mic or an arrhythmogenic effect, depending upon the level of membrane potential at which the slowing occurs. A broadening of the plateau phase at near-zero membrane potential, where both INa and Isi are inactivated in the course of the action potential and unable to recover from inactivation, will make the cell refractory against premature action potentials for as long as the membrane is kept at this depolarized level. If, however, the rate of repolarization is slowed at more negative levels of membrane potential (negative to about - 20 mV), spontaneuous action potentials often arise from the prolonged repolarization phase (Cranefield 1977). This is probably the most important cause 18 P. Honerj~iger of the arrhythmias produced by Na channel-gate toxins. The coupling interval and the shape of the triggered action potential depend on the recovery kinetics of INa and Isi during the after-depolarization. As shown by Gettes and Reuter (1974) this recovery is voltage dependent, the time constant for recovery OfVmax increasing from less than 20 ms at -80 mV to more than 100 ms at -60 mV. When the membrane is further depolarized to -9 mV, INa is completely inactivated and the time constant for recovery of" Isi ificreases from about 60 ms to about 250 ms. The critical toxin-induced prolongation of the repolarization phase which leads to re-excitation will therefore depend on the level of membrane potential at which the individual agent slows repolarization. In the case of aconitine (see following sections), which slows repolarization at about -60 mV, triggered activity occurs as soon as the action potential is prolonged by only about 30 ms (cat papillary muscle; Heistracher and Pillat 1962). In contrast, veratridine (see following sections), which prolongs the repolarization phase already at -20 mV, induces spontaneous activity after prolonging the action potential by more than 400 ms (guinea-pig papillary muscle; Honer]alger and Reiter 1975). After-depolarizations, which result from a delay of the repolarization process, have to be distinguished from oscillatory after-potentials, which occur after the membrane has repolarized completely (Bozler 1943; Ferrier 1977). The latter phenomenon is frequently observed in cardiac cells under conditions of Ca overload and in association with after-contractions (Reiter 1962; Kaufmann et al. 1963). Oscillatory after-potentials result from an oscillatory inward current that is thought to be triggered by a phasic release of Ca ions from an intracellular store (Kass et al. 1978a,b). Thus, an oscillatory Ca release from an intracellular store is regarded as the cause for both after-contractions and oscillatory after- potentials. Kass et al. (1978b) suggested that the oscillatory inward current may represent either the flow of cations through a non-selective ion channel or the inward movement of Na ions via an alectrogenic Na-Ca exchange mechanism activated by the rise in free intracellular Ca concentration. Although oscillatory after-potentials are inhibited by the specific Na channel blocker TTX (Kass et al. 1978b; Rosen and Danilo 1980), oscillatory after-potentials are not likely to be mediated by a delayed reopening of Na channels. The work by Kass et al. (1978b) suggests that any intervention which suppresses the intracellular Ca release responsible for the oscillatory after-potentials will also inhibit oscillatory after-poten- tins. TTX has a negative inotropic action (Sect. 4.2.5) and may thus inhibit oscillatory after-potentials indirectly by reducing intracellular Ca release. Cranefield (1977) has extended the term after-depolarization to include oscillatory after-potentials. He distinguishes between "early after-depolari- Cardioactive Substances that Prolong the Open State of Sodium Channels 19 zations", which interrupt the repolarization phase of the action potential, and "delayed after-depolarizations", which follow complete repolarization.

4.2 Ceveratrum Alkaloids

4. 2.1 Sinoatrial Node There are no reports on the effects of ceveratrum alkaloids on intracellular potentials in the sinoatrial node, but there is some information on the effects on atrial rate in the dog heart-lung preparation and in isolated guinea-pig atria. Benforado (1957) investigated the effects of nine cevera- trium ester alkaloids (veratridine, cevadine, veratroylzygadenine, vanillo- ylzygadenine, germbudine, neogermitrine, germitetrine, protoveratrine A, and protoveratrine B) on the dog heart-lung preparation by means of electrocardiographic (ECG) recording. Only cevadine produced a consistent chronotropic effect, i.e. a sinus tachycardia. The general lack of chronotropic activity shows clearly that the marked bradycardia seen following administration of ester alkaloids to the intact animal has no direct cardiac component (Benforado 1957). The mechanism of the positive chronotropic effect of cevadine is unknown. Cevine, the 3-~-hydroxyl isomer of the alkamine veracevine, causes a bradycardia in the dog heart- lung preparation (Moe and Krayer 1943) by an unknown mechanism. Germine-3-acetate (31-540 umol/litre) has a positive chronotropic effect on isolated guinea-pig atrium that is abolished by 0.5 t~mol/litre propranolol (Seifen 1969). This may indicate that germine-3-acetate affects the sinoatrial node by releasing catecholamines from intracardiac nerve fibres.

4.2.2 Atrium Horackova and Vassort (1973, 1974) analysed the effect of the alkaloid mixture veratrine (veratrine sulphate, Sigma) on small atrial bundles of the frog heart. They used the double sucrose-gap technique to record both membrane potential and membrane current under membrane-potential control. Veratrine prolongs the repolarization phase of the action potential, which agrees with the earlier findings that veratrine and one of its constituents, cevadine, prolong the action potential in isolated dog and rat atrial tissue (Brooks et al. 1955; Matsuda et al. 1953). The addition of TTX (3 umol/litre) completely suppresses the effect of veratrine on the action potential of frog atrial preparations. Under voltage-clamp conditions, veratrine prolongs the phase of inward current associated with moderate step depolarizations from the normal resting potential, and this induced inward current decays only slowly after repolarization 20 P. Honerj/iger

(inward "tail" current). The veratrine-induced inward current is abolished by TTX (3 pmol/litre), and veratrine fails to modify the remaining inward current (Isi) if external Na ions are replaced by sucrose. If Na ions are replaced by Li, veratrine increases inward current, as in the presence of external NA. These findings indicate that veratrine prolongs the open state of Na channels in frog atrial fibres. During a depolarizing pulse, the decay of INa is slowed by veratrine, indicating that the h gate closes more slowly in a fraction of Na channels. In the modified channels, the time constant of inactivation (rh) during depolarization by 40 mV from the normal resting potential is increased to 1.1-1.9 s (18°-20°C), i.e. by 3 orders of magnitude. There is no obvious effect on the activation of Na channels upon depolarization, but the removal of activation after repolarization is slowed, as indicated by the inward tail currents that decline exponentially with time constants of 0.4-0.7 s. This shows that veratrine has little or no effect on the rate constant am, but markedly reduces ~m 1. These effects are not identical to the effect of veratridine on Na channels in the frog node of Ranvier (Sect. 3.2), although in both types of cells, veratridine and veratrine, respectively, increase the Na inward current associated with the action potential. In the frog node of Ranvier, veratridine diminishes both a m and ~m, i.e, ~'m is increased during depolarization as well as after repolarizatio n 2. Furthermore, veratridine completely removes inactivation in the neural Na channels. It is not clear whether this discrepancy reflects a genuine difference 0etween neural and myocardial Na channels or whether the cardiac effects are due to ceveratrum alkaloids other than veratridine present in the alkaloidal mixture veratrine. Horackova and Vassort (1973, 1974) were the first to show that the effect on sarcolemmal PNa and the positive inotropic effect of veratrine are intimately linked. They found that the positive inotropic effect is abolished by TTX (3 umol/litre), which also blocks the veratrine-induced prolongation of the action potential. The positive inotropic effect of veratrine on frog atrial muscle develops during identical repetitive depolarizations under voltage-clamp conditions indicating that it is related to the change of membrane current and not to the veratrine-induced alteration of the duration of the action potential. As shown by Horackova and Vassort (1974) the veratrine4nduced Na inward current does not lead to an increase in the rate of force development of the simultaneous contraction within a given excitation-contraction cycle. In a previously resting

1 a m and /3m are the rate constants for the activation and deactivation, respectively, of the m gate of the Na channels (Hod~kin and Huxley 1952). These rate constants are related to the time constant of the m gate by the equation 7rn = 1/(am + ~m) 2 Ulbrieht (1969) uses the symbols c~s and/3 s for the veratridine-modified Na channels Cardioactive Substances that Prolong the Open State of Sodium Channels 21 veratrine-treated muscle, the positive inotropic effect appears only during the course of several excitations above a critical frequency. This shows that the bioelectric and positive inotropic effect of veratrine are linked by a slowly responding accumulative process (Sect. 4.2.5). All the findings suggest that veratrine increases the force of contraction by increasing the intracellular Na concentration through its effect on the Na channels. An intracellular accumulation of Na ions is expected to activate Na-Ca exchange (Chap. 5) which could explain the positive inotropic effect.

4.2. 3 A trioventricular Node

There are no reports on the effect of ceveratrum alkaloids on the transmembrane electrical activity of cells of the atrioventricular node. Using extracellular recording electrodes, Swain and McCarthy (1957) studied the effect of ceveratrum alkaloids on atrioventricular conduction in the dog heart-lung preparation. Veratrine, its main constituents veratridine and cevadine, and protoveratrine (a mixture of the protoveratrines A and B) each slow atrioventricular conduction. In the case of veratrine this effect is particularly pronounced at high frequencies, while at low frequencies veratrine may even accelerate atrioventricular conduction. At high frequencies the effect of veratrine is characterized by alternation of atrioventricular conduction time. The spontaneous firing rate of the atrioventricular node recorded after elimination of the activity of the sinoatrial node is enhanced by protoveratrine.

4. 2. 4 PurMn]e Fibres

Arbel et al. (1975) studied the effect of veratrine (Sigma) on the isolated ventricular conduction system of the dog heart with intracellular micro- electrodes. They found that superfusion of Purkinje fibres in the right bundle branch with solution containing 0.1-1 ~g/ml veratrine causes a slowing of repolarization that affects the plateau phase and terminal repolarization phase of the action potential. The latter effect may result in the appearance of a distinct after-depolarization. Higher veratrine concentrations induce oscillatory responses superimposed on the prolonged repolarization phase. The after-depolarization was followed in some experiments by a slight hyperpolarization. The prolonging effect on the repolarization phase is reversed by washout with veratrine-free solution. In ventricular muscle cells of the anterior papillary muscle dissected together with the bundle branch, the prolongation of the action potential by veratrine is less, both in absolute units and if related to the control duration, than that recorded simultaneouSly from Purkinje fibres. 22 P. Honerj~iger

The effect on repolarization is interpreted as resulting from the persistent activation of Na channels because it is inhibited by TTX (0.3-3 ~mol/ litre). Arbel et al. (1975) note that TTX abolished the effect of veratrine on repolarization at a time when the Purkinje fibre is still excitable, indicating that at least a major fraction of the normal Na channels that mediate the upstroke of the action potential is not blocked by that concentration of TTX. One might thus conclude that the Na channels activated by veratrine are more sensitive to TTX than normal Na channels. In this context it is of interest that even during the normal action potential of Purkinje fibres, a component of persistent and TTX-sensitive Na current was detected (Attwell et al. 1979); this explains why TTX has a marked shortening effect on the normal action potential of Purkinje fibres (Cora- boeuf et al. 1979). It appears that, in the absence (Coraboeuf et al. 1979) as well as in the presence of veratrine (Arbel et al. 1975), the effect of TTX on the repolarization phase requires lower concentrations than its inhibitory effect on the rate of rise of the action potential. As pointed out by A ttwell et al. (1979), such a differential sensitivity does not necessarily indicate that the Na channels activated during upstroke of the action potential and those activated during the plateau phase differ in their sensitivity to TTX. Rather, the blockade of the same small fraction of Na channels by TTX may result in a larger effect on action potential duration than on upstroke velocity, if the small fraction of the Na current blocked by TTX is a large fraction of the net inward membrane current flowing during the plateau phase. Thus, the different sensitivity to TTX of the two phases of the action potential might be due to the fact that the upstroke is almost entirely the result of net inward Na current through Na channels, whereas during the plateau a considerable fraction of the inward ionic current is balanced by outward K current. This considera- tion applies equally to the situation when the action potential is prolonged by Na channel-gate toxins. It is therefore not necessary to postulate that veratrine activates an additional population of Na channels differing by an abnormally high sensitivity to TTX from normal Na channels responsible for the upstroke of the action potential. In the frog node of Ranvier, normal and veratridine-modified Na channels were identical in their sensitivity to TTX (Ulbricht 1974). Veratridine (0.1 umol/litre) has recently been shown to increase the force of contraction of canine Purkinje fibres stimulated at 1 Hz (Vassalle and Bhattacharyya 1980). The positive inotropic effect, as well as the prolongation of the action potential observed in the same study, are antagonized by the local anaesthetics procaine or benzocaine. Cardioactive Substances that Prolong the Open State of Sodium Channels 23

4.25 Ventricular Muscle Although the direct positive inotropic effect of ceveratrum alkaloids had already been discovered at the beginning of this century (see Krayer and Acheson 1946), analytical and quantitative studies of this effect were performed only in recent years (Reiter 1963; Koch-Weser 1966; Horackova and Vassort 1973, 1974; Honerjdger and Reiter 1975, 1977a; Honerjdger 1977, 1980). An understanding of the cardiac effects of these alkaloids was only possible after Ulbricht (1969) had shown that veratridine alters the Na channels of the excitable membrane. Detailed information on the effects of individual ceveratrum alkaloids on ventricular myocardium was obtained in the isolated isometrically contracting papillary muscle of the guinea pig (Honerj~iger and Reiter 1975, 1977a;Honerjdger 1977, 1980). In this section, various aspects of the positive inotropic effect are described including the concentration-effect relationships, the modifications of individual phases of the isometric contraction curve and the differentiation of the direct from a neurally mediated positive inotropic effect. Effects on transmembrane electrical activity are also described. Finally, the mechanism of positive inotropic action is discussed, as well as arguments suggesting that the positive inotropic effect is an indirect consequence of the modification of sarcolemmal Na channels leading to increased Na influx, intracellular Na accumulation and increased Na-Ca exchange. Positive Inotropic Effect The positive inotropic effect of ceveratrum alkaloids on the isometrically suspended guinea-pig papillary muscle results from an increase in the rate of force development, while the time to peak force is unaltered or slightly shortened (Figs. 2, 3). The term klinotropic was introduced to denote influences on the steepness of the contraction curve (Bohnenkamp 1922; Reiter 1972a). The positive inotropic effect of ceveratrum alkaloids is thus of the positive klinotropic type. With respect to their influence on the relaxation phase, two types of effect can be distinguished. Veratri- dine (Fig. 2), as well as cevadine (Reiter 1963; Honerjgiger 1977), veracevine, zygadenine, and veratroylzygadenine (Honerjdger 1977)markedly prolong relaxation time and thereby total contraction time. In contrast, no major effect on total contraction time is observed under the influence of germitrine (Fig. 3), other germine di- and triesters or the protoveratrines A and B (Honerjdger 1977). This varied effect on the relaxation phase is probably due to the different modifications of the repolarization phase by these alkaloids. Furthermore, TTX inhibits the effect of veratridine on relaxation in addition to that on the repolarization phase suggesting a causal connection (see Fig. 9). A direct effect on the rate of Ca sequestration by sarcoplasmic reticulum thus seems to be ruled out. 24 P. Honerj~iger

/ 1.6 pM (F)20 ~0.8 pM '~ 1-6HM

°NTR°' 0L/ I I I I 200 ms 200 ms

Fig. 2. The positive inotropic effect of veratridine. Isometric contraction records from an isolated guinea-pig papillary muscle contracting at 1 Hz and exposed to cumulatively increasing concentrations of veratridine. Note the prolongation of the relaxation phase. (From Honerjiiger and Reiter 1975)

80 nM 2.0 40 nM F c 60 nM (g) 20 nM 1.5 ~1 10 0. Fig. 3. The positive inotropic effect of germitrine. Isometric contraction records 1.0 /Z///~ C502T~O L from an isolated guinea-pig papillary muscle contracting at 1 Hz and exposed to ;Yf \\ cumulatively increasing concentrations of 0.5 germitrine. Note that this ceveratrum alkaloid does not markedly alter the dura- f 'M_ tion of contraction. (From Honerf~'ger and i ! Reiter 1977a, by permission of American 200 ms Heart Association)

Indeed, veratridine (1 #mol/1 to 1 mmol/1) is reportedly without effect on Ca uptake by sarcoplasmic reticulum isolated from rabbit skeletal muscle (Johnson and Inesi 1969). The positive inotropic activity resides in the alkamine part of the ceveratrum alkaloids, but esterification increases the potency. The alkamines zygadenine and veracevine (each at 0.5 mmol/litre) produce a positive inotrpic effect, while germine (0.8 mmol/litre) and protoverine (5 mmol/ litre) are inactive (Honer]iiger 1977). Graded, steady-state positive inotropic effects can be obtained with different concentrations of ceveratrum alkaloids, suggesting a reversible binding reaction with the receptor for the positive inotropic effect. In fact, the positive inotropic effect of Cardioactive Substances that Prolong the Open State 6f Sodium Channels 25

Positive inotropic effect,AF c (% of maximum)

100 I I I

','-0 30 Y 20 10 / !1 I I I I I 010-9 10-8 10 -7 10-6 tO-5 tO-4 10-3 Alkaloid concentration (mol/I)

Fig. 4. Concentration-effect relationships for the positive inotropic effect of ceveratrum ester alkaloids: Abscissa, molar concentration of alkaloid, logarithmic scale; ordinate, positive inotropic effect expressed as percent of the maximum increase in force of contraction obtained with each alkaloid. Means of at least six guinea-pig papillary muscles for each alkaloid (SEM < 7%).Germitetrine (1), protoveratrine A (2), germitrine (3), protoveratrine B (4), neogermitrine (5), desacetylgermitetrine (6), germerine (7), germbudine (8), veratroylzygadenine (9), neogermbudine (10), veratridine (11), cevadine (12), zygacine (13), and germine-3-acetate (14). (From Honer- ]iiger 1977)

veratridine disappears during washout with alkaloid-free medium, although only very slowly (Honer]dger and Reiter 1975). The concentration-dependence of the positive inotropic effect obtained with 14 different ceveratrum ester alkaloids is shown in Fig. 4. The absolute maximum increase in force of contraction, corrected for cross-sectional area of individual muscles, does not differ significantly among the various alkaloids. The concentration-effect relationships are nearly parallel and relatively steep. Each curve extends over approximately 1.5 logarithmic units. The concentration-effect relationship for the increase of PNa by veratridine is less steep and compatible with a reversible one-to-one binding reaction between veratridine molecules and the receptor at the Na channel (Carte- rall 1975). Presumably, the concentration-effect curve for the positive inotropic effect of ceveratrum alkaloids is limited by the capacity of the muscle to increase force development and not by the ability of ceveratrum alkaloids to increase sarcolemmal PNa (Honer]dger and Reiter 1975, 1977a). The maximum positive inotropic effect of veratridine and 26 P. Honerj~iger germitrine was found to be 68% and 81%, respectively, of that of the cardioactive steroid dihydro-ouabain tested on the same papillary muscle (Honerjdger and Reiter 1975, 1977a). Like other depolarizing interventions, veratridine causes a release of noradrenaline from intracardiac adrenergic nerve endings (Moss et al. 1974). The release is mainly of the exocytotic type (Thoa et al. 1975; Ross and Kelder 1979). In addition, veratridine has a TTX-resistant, reserpine-like noradrenaline-depleting effect on the storage vesicles, but this effect does not normally lead to increased release of noradrenaline from the nerve ending (Bdnisch et al. 1980). The positive inotropic effect of ceveratrum alkaloids, including veratridine, persists in muscles that have been depleted of noradrenaline by reserpine pretreatment (I£och-Weser 1966; Honerjiiger and Reiter 1975, 1977a). This argues against the participation of a neural mechanism in the positive inotropic action under normal conditions (1 Hz contraction frequency). In contrast, concentrations of veratridine or cevadine exceeding those maximally effective at a frequency of 1 Hz increase the force of the rested-state contraction entirely by a neurally mediated mechanism (floner]iiger 1980). This positive inotropic effect is absent in the presence of a neuroactive, but not yet cardioactive concentration of TTX (100 nmol/litre) and in muscles from reserpine-pretreated animals. It is inhibited by 50 nmol/litre (-)propranolol, but not by the same concentration of the dextrorotatory enan- tiomer. The concentration-effect relationships for the direct and the neurally mediated positive inotropic effect of veratridine are illustrated in Fig. 5. While all these findings demonstrate that ceveratrum alkaloids affect isolated ventricular mammalian myocardium normally by a direct myotropic action, Basseches and Bianchi (1976) conclude, on the basis of their experiments on the perfused frog ventricle, that veratridine acts by enhancing transmitter release from cholinergic and adrenergic nerve fibres. The discrepancy may in part be due to the fact that Basseches and Bianchi (1976) used a field stimulus on their preparations which also excited both cholinergic and adrenergic nerve terminals. Under these conditions the frog ventricle may be more sensitive to the neural effects of veratridine.

Transrnembrane Electrical Activity Veratridine prolongs the repolarization phase of the action potential in guinea-pig papillary muscle (Honerjiiger and Reiter 1975) and the effect of a relatively high concentration (2 umol/litre) is illustrated in Fig. 6. For this experiment the papillary muscle was stimulated only once every 5 rain which, because of the frequency-dependence of the inotropic action, prevents the positive inotropic effect of veratridine. Secondary superimposed effects on the action potential expected to result from Cardioactive Substances that Prolong the Open State of Sodium Channels 27

25 I I i i I I | dEc 2O Contractions at 1Hz ~/T~ t 10 /÷ .e2.,a, contraction / (9)

, 0.1 0.3 1.0 3.0 1() 310 Veratridine concentration (pmoFI) Fig. 5. Concentration-effect relationships for the direct (left curve) and neurally mediated (right curve) positive inotropic effect of veratridine on guinea-pig papillary muscle. The direct (reserpine-insensitive) effect was measured on muscles contracting at 1 Hz and exposed to cumulatively increasing concentrations. The presynaptic (reserpine-sensitive) effect was obtained on muscles that were incubated at rest with a single concentration of veratridine to determine its effect on the subsequent rested- state contraction. Number of muscles is given in parentheses. The inset shows a control rested-state contraction and a second one amplified by exposure of the resting muscle to 30/lmol/litre veratridine (V); calibration: 5 mN (vertical), 0.2 s (horizon- tal). (From Honerja'ger 1980)

VERATRIDINE NO DRUG TETRODOTOXIN

200[ ["1-8 I 20 q ms 200

Fig. 6 A--C. Effect of veratridine on the transmembrane action potential of guinea- pig papillary muscle and blockade of the effect by TTX. Before (A), 25 min after addition of 2 #mol/litre veratridine (B), and 7 rain after additonal application of TTX (10 #mol/litre) 1(C). Continuous microelectrode impalement. Stimulation frequency was 0.2 min- . The broad trace is the differentiation of the upstroke of the action potential and was obtained at the faster sweep speed. (From Honerjiz'ger 1977)

increased intracellular Ca, notably an abbreviation of the action potential (Isenberg 1975; Bassingthwaighte et al. 1976), are hence avoided. Vera- tridine does not affect the resting membrane potential or Vma x (Fig. 6). The overshoot of the action potential is slightly reduced (by 4 mV). The plateau phase assumes an upward concave contour in contrast to the con- vex shape present under control conditions. The repolarization phase is 28 P. Honerj~iger slowed particularly as it proceeds from +16 mV to 20 mV and from -60 mV to the resting potential level. Additional application of TTX (10 gmol/litre) completely restores the original action potential duration while reducing Vmax by about 50% (Fig. 6C). The sensitivity of the veratridine effect to TTX strongly suggests that it is mediated by Na channels. As discussed in Sect. 4.2.4, the stronger effect of TTX on repolarization than on Vmax does not argue against the identity of the Na channels responsible for the upstroke with those activated by veratridine during the plateau phase. A prolongation of the cardiac action potential may lead to an extracellular accumulation of K ions resulting in an after- depolarization (Cleeman and Morad 1976). In the presence of veratridine such an effect may play an additional role during the final phase of repolarization. Reiter (1963) pointed out that the monoesteralkaloids veratridine and cevadine differ in their effect on the repolarization phase. Cevadine prolongs the plateau phase of the action potential whereas veratridine predominantly slows the subsequent phase of normally fast repolarization. This difference is reflected by the relaxation phase of the isometric contraction. Cevadine slows the initial part and veratridine delays the final part of relaxation (Reiter 1963). The effect of the germine triester germitrine on action potential differs from that of veratridine in that only the very final phase of repolarization is prolonged, causing the appearance of an after-depolarization lasting longer than 1 min (Honerj~ger and Reiter 1977a). As shown in Fig. 7, germitrine stops the repolarization process at a level about 6 mV positive to the resting potential. Thereafter the membrane depolarizes again slightly, to a maximum level about 9 mV positive to the resting potential, and then repolarizes very slowly. The original level of the resting potential is eventually reached after a slight hyperpolarization (Fig. 8). The after-depolarization produced by di- and triesters of germine, as well as by the two tetraesters of protoverine, protoveratrine A and protoveratrine B (Fig. 8) must be distinguished from the oscillatory after-potentials observed under conditions of Ca overload. The latter occur after complete repolarization and in an oscillatory manner: They are of shorter duration and not mediated by Na channels (Sect. 4.1.2). The germitrine- induced after-depolarization is completely and reversibly blocked by 10 ~mol/litre TTX, suggesting that it results from a delayed activation of Na channels (Honer]alger and Reiter 1977a). The amplitude of the after- depolarization increases with the concentration of germitrine. The demonstration of the germitrine effect requires a very low stimulation frequency (cf. Fig. 7). If a germitrine-treated muscle is stimulated repetitively at intervals shorter than the duration of the germitrine- induced after-depolarization, the resting potential shows a stepwise Cardioactive Substances that Prolong the Open State of Sodium Channels 29

a b

mV 0

0.2gE -80 II 100 ms/1 ms

10 mV

I ,I 0.5 min

Fig. 7 a,b. Effect of germitrine on the transmembrane action potential and contraction of a guinea-pig papillary muscle stimulated once every 10 rain. The upper panels show the action potential and isometric contraction at a slow sweep speed and the upstroke of the action potential at a fast sweep speed. The records shown in the lower panels were obtained simultaneously with the corresponding upper records on a second oscilloscope at higher gain and very slow sweep speed. They show the membrane potential before and during a period of 1.5 min after the action potential. The action potential causes the interruption (arrow) of these traces. Records were obtained before (a) and 74 rain after (b) addition of 2/2mol/litre germitrine. Note incomplete repolarization (b, upper panel), which gives rise to an after-depolarization (b, lower panel). (From Honerja'ger and Reiter 1977a, by permission of American Heart Asso- ciation) decrease (Honer/alger and Reiter 1977a). This phenomenon is analogous to the well-known "summation" of after-depolarizations observed ifvera- tridine-treated nerve fibres are subjected to high-frequency stimulation (Ulbricht 1969). In the presence of high germitrine concentrations, the repetitive stimulation leads eventually to arrhythmic activity of the papillary muscle if the after-depolarization reaches the threshold to eficit an action potential. At 80 nmol/litre germitrine, the maximally effective inotropic and non-arrhythmic concentration at 1 Hz frequency, the summation of after-depolarizations results in a decrease of the resting potential by about 10 mV in the steady-state (Honer]alger and Reiter 1977a). Mechanism of the Positive Inotropic Effect Studies of the effects of veratridine (Reiter 1963; Honerjdger and Reiter 1975) and germitrine (Honer]ager and Reiter 1977a) revealed a close association of the bioelectric and inotropic effects in guinea-pig papillary muscle that strongly suggests a causal relationship. The results may be summarized as follows: 30 P. Honerj~iger

I__~ No drug

Germerine

E~- Neogermbudine

E~ Germbudine E_ Desacetylgermitetfine

[_ Neogermitrine

~ermitrine Em E_ ~. ProtoveratrineA [_ ~ rotoveratrine B ~~=~..._.~ Germitetrine E_ I I I I 1 2 3 4 min

Fig. 8. After-depolarizations induced in guinea-pig papillary muscle by various ceveratrum ester alkaloids. High-gain records of transmembrane potential immediately before and after the stimulation of an action potential. The action potential causes the interruption of the traces at the time marked 0. Experimental procedure as in Fig. 7. The following alkaloid concentrations were used (/~mol/litre): germerine (4); neogermbudine (10); germbudine (8); desacetylgermitetrine (4); neogermitrine (2); germitrine (2); protoveratfine A (3); protoveratrine B (12); and germitetrine (4). Vertical calibration was 5 mV. (From Honer]alger 1977)

1. Inotropically effective concentrations of veratridine or germitrine also produce changes of the transmembrane electrical activity attributable to an increased PNa" 2. TTX inhibits the bioelectric as well as the positive inotropic effect of veratridine or germitfine. 3. While the bioelectric and inotropic effects appear to occur simultaneously in repetitively stimulated preparations, a closer analysis reveals that the positive inotropic effect is in fact delayed with respect to the phase of increased PNa produced by either veratridine (Honer]alger 1977) or germitrine (Honer]alger and Reiter 1977a)and decays slowly in the non-excited muscle. Cardioactive Substances that Prolong the Open State of Sodium Channels 31

4. As a consequence of the delayed and transient inotropic response to the alkaloid-induced PNa' the positive inotropic effect does not manifest itself when contractions are separated by long rest intervals (e.g., 5 min). The inotropic effect appears and grows as the frequency is increased from 0.004 Hz to 0.5 Hz. 5. The positive inotropic response to the combination of a ceveratrum alkaloid with dihydro-ouabain is much larger than the sum of the responses to either drug alone. This synergism points to the importance of the intracellular Na concentration in mediating the positive inotropic effect of ceveratrum alkaloids. The extent of the alkaloid- induced increase in [Nail must be enhanced by an additional inhibition of the Na pump. TTX has proved a valuable tool in identifying the mechanism of the positive inotropic action of ceveratrum alkaloids and Na channel-gate toxins in general. The selective action of TTX in blocking Na channels of excitable membranes at an extracellular membrane site has been extensively documented (see Narahashi 1974). While suppressing INa, TTX does not significantly affect the slow inward current in frog atrial myocardium (Tarr 1971) or, at 47 gmol/litre, in dog ventricular myocardium (Beeler and Reuter 1970b). With regard to the analysis of changes in contractile activity it is important to note that TTX does not appear to penetrate through the plasmalemma as shown in studies on the squid giant axon (Narahashi et al. 1966; Ro]as and Rudy 1976). Thus, TTX is unlikely to interfere directly with any intracellular process of excitation- contraction coupling or the contraction of the contractile proteins. TTX may affect myocardial force of contraction by two recognized mechanisms 3. It abolishes the positive inotropic effect associated with field stimulation by blocking the excitability of intracardiac adrenergic nerve fibres (Feinstein and Paimre 1968). This effect requires very low concentrations (31-63 nmol/litre) that have virtually no effect on the Na channels of guinea-pig myocardial ceils as judged by the upstroke of the action potential (Baer et al. 1976). Similarly low concentrations of TTX likewise abolish the presynaptic (or "indirect") positive inotropic effect of veratridine or cevadine (see p. 26). The second mechanism by which TTX affects myocardial force involves the blockade of sarcolemmal Na channels. At a frequency of 1 Hz, 10 #mol/litre TTX reduces 9max by about 50% and peak force of contraction by about 30% in noradrenaline- depleted guinea-pig papillary muscle (Honerjiiger and Reiter 1975). This direct negative inotropic action has to be taken into account when TTX is used as an antagonist of positive inotropic agents. 3 The citrate buffer present in the commercial preparation of TTX does not cause a significantinotropic effect of its own when tested at a concentration corresponding to 15/~mol/litre TTX (Honer]iiger and Reiter 1975) 32 P. Honerj~iger

The antagonistic effect of TTX against both the bioelectric and inotropic effects of veratridine is illustrated in Fig. 9. TTX completely and reversibly abolishes the positive inotropic effect of veratridine. The negative inotropic effect of TTX in the presence of veratridine is much larger than that observed in its absence, showing the specific nature of the antagonism. As Fig. 10 shows, TTX antagonizes the positive inotropic effect of ceveratrum alkaloids selectively. The effects of represen- tatives of three other classes of inotropic agents, a cardioactive steroid, a catecholamine and a methylxanthine are not, or much less, inhibited by TTX. Interestingly, the positive inotropic effect of dihydro-ouabain, which also depends critically on Na influx (Reiter 1972a), is less sensitive to TTX than that of the ceveratrum alkaloids veratridine, cevadine, and germitrine. In the case of the steroid, the increase in intracellular Na concentration results from inhibition of the Na pump, and the extent of this increase is diminished if passive Na influx is inhibited by TTX. This might explain the slight antagonism observed (Fig. 10). TTX is expected to be more effective in reducing the alkaloid-induced Na influx than the normal Na influx, on which the inotropic action of the steroid may depend, for the following reasons: Firstly, the normal Na influx probably occurs through TTX-sensitive Na channels as well as through TTX-insensitive ion channels such as slow (Ca, Na) channels (Reuter and Scholz 1977). Secondly, the Na influx induced by Na channel-gate toxins during the repolarization phase is probably more strongly inhibited by TTX than the Na influx occurring during the upstroke, as suggested by the higher TTX sensitivity of the repolarization phase (Sect. 4.2.5).

l~-Veratridine, 1.6 FN I Control TTX, 16 I~H

mV \ 0 \ \ / " \ I, t.0g -100 l

I I I 0 200 &00 msec Fig. 9. Reversible inhibition of the effects of veratridine on action potential and contraction by TTX in guinea-pig papillary muscle. The records were obtained (from left to right) before drug application, 50 min after addition of veratridine ( 1.6 gmol/litre), 5 rain after additonal application of TTX (16/~mol/litre), and after washout of TTX during maintained exposure to veratridine. Muscle was from a reserpine-pretreated animal. Contraction frequency was 1 Hz. (From Honer]iiger and Reiter 1975) Cardioactive Substances that Prolong the Open State of Sodium Channels 33

i 100r CEVADINE "~ I- VERATRIDINE /° -'~ // ,/),jJ /"

~G ® GERMITRINE /°_ //')/"/'~ el [/TX] = 0 2 [TTX] =10 pM > / i"sl,]f no DIHYDRO-OUABAIN ,0.~/o

NORADRENALIN~

5 I THEOPHYLLINEZ/JJ ~ {raN)

0 L i i i 4.1 1 10 100 x EC50

Fig. 10. Specificity of TTX as an antagonist of inotropic drugs. Concentration-effect relationships obtained in the absence and presence of TTX (10//mol/litre) by cumu- lative drug additions. Ordinate: positive inotropic effect expressed as percent of the maximum effect obtainable under each condition, except in the case of theophylline where the absolute increase of force of contraction is given; abscissa: drug concentration in logarithmic scale expressed in units of half-maximally effective concentrations in the absence of TTX. Means -+ SEM {vertical bars] of at least six guinea-pig papillary muscles. For the ceveratrum alkaloids (cevadine, veratridine, and germitrine) control and TTX experiments were done on separate muscles. Each of the other drugs was tested, both in the absence and presence of TTX on the same muscle. Contraction frequency was 1 Hz. (From Honer]iiger 1977) 34 P. Honerj~iger

The inhibitory effect of 10 tsmol/litre TTX is fully surmountable by increases in the concentration of the ceveratrum alkaloids (Fig. 10). This should not be taken as evidence for a competitive type of antagonism between the alkaloids and TTX. The antagonism between Na channel-gate toxins and TTX at the level of Na channels is non-competitive (Sect. 3.10.1). The inotropically effective range of alkaloid concentrations probably modifies only a small fraction of Na channels, and this fraction can still be affected by the gate toxins at a TTX concentration (10/~mol/litre) that blocks only about 50% of the Na channels (Honer- ]iiger and Reiter 1975). Although the inhibition of veratridine effects by TTX suggests a causal relationship between the bioelectric and the inotropic effect of veratridine, it remains uncertain whether veratridine increases force of contraction by increasing Na influx or by prolonging the repolarization phase (thereby indirectly prolonging the phase of Ca influx through slow channels). However, the prolongation of the action potential, under the conditions of Fig. 9, is restricted to negative levels of membrane potential where a prolongation of repolarization per se produces little positive inotropic effect (Wood et al. 1969). In the case of germitrine the positive inotropic effect is even associated with a marked shortening of the action potential duration, in addition to the reduction of the resting membrane potential reflecting the increased PNa (Honer]iiger and Reiter 1977a). This rules out a secondary effect on Isi as a cause of the positive inotropic effect for this ceveratrum alkaloid. The strongest evidence in support of the hypothesis that ceveratrum alkaloids act by increasing inward Na current is provided by the voltage-clamp experiments of Horackova and Vassort (1973, 1974) on frog atrial muscle (Sect. 4.2.2). The germitrine4nduced after-depolarization is particularly suitable for a study of the temporal relationship between the phase of increased PNa and the positive inotropic effect. By choosing an appropriate alkaloid concentration, the amplitude of the after-depolarization can be adjusted to a level which is well below the activation threshold for Isi (- 40 mV; New and Trautwein 1972) and which leaves the muscle electrically excitable throughout the course of the after-depolarization. Test contractions elicited at various times during or after the alkaloid-induced after-depolarization (Fig. 11) reveal a phase during which the ability of the muscle to develop force is increased. This positive inotropic influence reaches its maximum 1 min after the conditioning stimulus and thereafter decays with a half-life of 2.5 min. In contrast, the after-depolarization attains a peak value about 10 s after the conditioning action potential and decays with a half-life of 32 s. Thus the phase of increased PNa clearly precedes the phase of increased contractility. This is compatible with a role of the intracellular Na concentration in mediating the positive inotropic effect. Cardioactive Substances that Prolong the Open State of Sodium Channels 35

100 "ec l,oor.~

][ t\ INOTROPIC ~E TER 50 DE POL;RIZATION ~ECT (t,Fe/ , "=:t 200 ms ]E

IL o o °

I I i I I I --~- 1 2 3 4 5 6 7 8 g 10 ; -: TIME AFTER ACTION-POTENTIAL UPSTROKE (rain) o----o REST INTERVAL AFTER RESTED-STATE CONTRACTION

Fig. 11. Temporal relationship between the germitrine-induced after-depolarization and the germitrine-induced positive inotropic effect as determined by single test contractions during and after the after-depolarization. Ordinate: membrane potential during the after-depolarization which followed an action potential elicited at 0 time ( 100% = 8.1 -+ 0.4 mV) (o) and peak force of test contraction minus peak force of prior rested- state contraction (100% = 79 + 16 rag) (o). The time-course of the after-depolarization is shown by means + SEM of 27-29 records obtained from six muscles incubated with 2 #mol/litre germitrine for at least 1 h. The positive inotropic effect (means + SEM of six to eight muscles) was determined in different muscles. Inset shows enhancement of test contraction (trace 2) elicited 1 rain after the conditioning rested-state contraction (trace I), i.e. during the falling phase of the after-depolarization. (From Honer]~'ger and Reiter 1977a, by permission of American Heart Association)

[Na]i will continue to rise during the period of increased PNa and sub: sequently decline owing to the activity of the Na pump. Direct verifica- tion of this point by the use of an intracellular Na-sensitive micro-elec- trode would be of great interest. It is generally accepted that the contraction of cardiac cells is directly linked to the increase in free Ca concentration of the myoplasm. An increased intracellular Na concentration can mediate a positive inotropic effect indirectly by increasing the transsarcolemmal influx of Ca ions, thereby loading cellular Ca stores that release Ca during contraction. If Na-Ca exchange is enhanced by the depolarization produced by the action potential (Sect. 5.3.4; Mullins 1979), an increased level of [Na]i may not only increase the steady influx of Ca ions but especially that occurring during the action potential. The evidence suggesting the existence of a Na-Ca exchange mechanism in the plasmalemma is discussed in Chap. 5. 36 P. Honerj~iger

Few attempts have been made to rule out alternative mechanisms of the positive inotropic effect by direct biochemical experiments. The activity of Na, K-ATPase isolated from guinea-pig hearts is not affected by concentrations of ceveratrum alkaloids (veratridine, cevadine, protoveratrine A and B, germitetrine, desacetylgermitetrine) that are much higher than those causing a maximum positive inotropic effect on guinea-pig papillary muscle (Portius and Repke 1964). Veratridine, cevadine, and protoveratrine lack an inhibitory effect on the active Na-K transport of erythrocytes (Kahn and Acheson 1955). Sperelakis and Pappano (1969) noted that veratrine did not significantly change the internal K and Na content of intact embryonic chick hearts during 15 min, which argues against inhibition of the Na pump. There is apparently no report on whether inotropically effective concentrations of ceveratrum alkaloids influence the concentration of cyclic nucleotides in myocardial cells. However, it should be noted that many functional characteristics of the inotropic action of ceveratrum alkaloids (and other Na channel-gate toxins) differ clearly from those associated with agents thought to act by increasing the cellular content of cyclic adenosine 3',5'-monophosphate (cAMP) (Sect. 5.1).

4. 2. 6 Cardiac Cells in Culture

During the embryonic development of chick and rat hearts, Vmax of the cardiac action potential and the susceptibility of action potential and contraction to TTX were shown to increase progressively (Bernard and Gargouil 1968; Ishirna 1968; Shigenobu and Sperelakis 1971; Sperelakis and Shigenobu 1972; Pappano 1972; McDonald et al. 1972; DeHaan et al. 1975; McDonald and Sachs 1975; Ii]ima and Pappano 1979). Thus, in the early embryonic stage (2-4 days in ovo), cardiac cells seem to lack fast Na channels. The action potential is mainly due to the opening of a slow Na channel insensitive to TTX (Sperelakis and Shigenobu 1972). Cardiac cells cultured in the form of monolayers invariably display TTX- insensitive action potentials even if the cells are taken from embryos older than 5 days. Cultured in aggregates, they generally retain the TTX sensi- itivity of the embryonic heart from which they were derived (McDonald et al. 1972;McLean and Sperelakis 1976). Sperelakis and Pappano (1969) investigated the effects of veratrine and veratridine on ventricular myoblasts obtained from 7-15-day chick embryos, but grown in monolayers. Veratridine (1.5-15 ~mol/litre) depolarizes the cells within a few minutes to membrane potentials of about -12 mV, and action potentials and beating cease. Before the depolarization begins, the repolarization phase of the action potential is prolonged by veratridine. TTX (63 ~mol/litre), although without effect Cardioactive Substances that Prolong the Open State of Sodium Channels 37 on action potential magnitude or Vmax, prevents the depolarization and prolongation of the action potential by veratridine. The depolarization by veratridine also occurs in Na-free Li solution. Veratridine reduces the membrane resistance. This is not prevented by TTX and is attributed to an increased K permeability. In 3- and 16-day aggregates of chick embryonic heart cells, veratridine produces depolarization and blocks the electrical and mechanical activities at concentrations exceeding 10 ~mol/litre (Romey et al. 1980). Romey et al. (1980) suggest that, like other Na channel-gate toxins, veratridine reveals Na channels that are normally in a silent form in monolayers or 3-day aggregates. The activation of Na channels by veratridine leads to an increase of Na influx into the myoblasts as determined with 22Na (Fosset et al. 1977). The effect is inhibited by TTX, the half-maximally effective concentration being 6.6 nmol/litre at a veratridine concentration of 22 ~mol/litre (Fosset et al. 1977). This degree of TTX sensitivity is similar to that of Vmax in cells at later stages of embryonic development (McDonald et al. 1972; Romey et al. 1980), but much higher than that of Na channels in adult guinea-pig ventricular myocardium (Baer et al. 1976; see also Fig. 6). Veratridine increases the influx of Ca ions in addition to that of Na ions (Sect. 5.3.2). Protoveratrine B also increases the Na uptake by cardiac cells of the chick embryo (Couraud et al. 1976).

4.3 Batrachotoxin

Batrachotoxin belongs to the most toxic substances known. Its lethal effect on mammals, which occurs after parenteral administration of less than 1 pg/kg or 1.86 nmol/kg, appears to be mainly due to its cardiac arrhythmogenic action (Daly and Witkop 1971). A study on anaesthetized rabbits showed that batrachotoxin produces a variety ofventricular arryth- mias, in addition to complete block of atrioventricular conduction (Kay- aalp et al. 1970). Predominant effects are the activation of multifocal ventricular ectopic beats interrupted by transient ventricular fibrillation or tachycardia. The terminal event is ventricular fibrillation.

4.3.1 Purkin]eFibres In spontaneously active Purkinje fibres isolated from the dog heart, the initial effect of batrachotoxin on the action potential is a prolongation of the final phase of repolarization (Hogan and Albuquerque 1971). It occurs at the lowest tested concentration of 1.9 nmol/litre at a time when there are no changes in the maximum diastolic membrane potential, Vmax, overshoot of the action potential, or rate of spontaneous diastolic depolarization. With increasing time of exposure to the toxin, the delay 38 P. Honerj~iger of repolarization progresses until the membrane fails to repolarize completely during the diastolic interval. At this stage the upstroke velocity and overshoot of the action potential are depressed, as expected from the inactivating influence of depolarization. The membrane potential of fibres depolarized by batrachotoxin is restored if the external Na concentration is reduced to 1 mmol/litre. At a concentration of 4.7 ~mol/ litre, TTX prevents the effect of batrachotoxin (1.9 nmol/litre) on the repolarization phase for as long as 30 min. These results clearly indicate the involvement of Na channels in the effect of batrachotoxin on the Purkinje fibre membrane. [logan and Albuquerque ( 1971 ) point out that "TTX did not block the action potential of the Purkinje fiber in a concentration which antagonized BTX [batrachotoxin] action". According to these authors, "resting sodium channels are opened by BTX" and "resting sodium channels in the electrogenic membrane may be functionally different from the sodium channels for membrane excitation". The TTX concentration used (4.7 ~mol/litre) causes a partial inhibition of Vmax in dog Purkinje fibres (approx. 50%, Coraboeuf et al. 1979). That this concentration causes complete inhibition of the effect of batrachotoxin, as opposed to partial inhibition of Vmax, does not argue against the identity of the Na channels involved in generating the action potential with those modified by batrachotoxin, as discussed in connection with the effect of veratrine on dog Purkinje fibres (Sect. 4.2.4). Hogan and Albuquerque (1971) state that "the same sequence of alterations produced by BTX on the Purkinje fiber membrane was observed whether or not the tissue was being driven". Since their preparations were reportedly spontaneously active, this does not rule out a dependency of the effect of batrachotoxin on electrical activity. Such a dependency was reported to exist in the eel electroplaque (Bartels-Bernal et al. 1977), frog node of Ranvier (Sect. 3.3), and guinea-pig papillary muscle (Sect. 4.3.2).

4.3.2 VentricularMuscle similar to its effect on Purkinje fibres, batrachotoxin prolongs the repolarization phase of the action potential in guinea-pig muscle, and this effect is inhibited by TTX (Honer]iiger and Reiter 1977b). The results indicate that batrachotoxin causes its effect on the ventricular action potential by selectively prolonging the open state of Na channels. At low concentrations of the alkaloid (0.75-60 nmol/litre), the effect on repolarization occurs only after a series of conditioning action potentials. The first action potential after addition of batrachotoxin is unaltered even after prolonged exposure of the resting muscle to the toxin. During repetitive stimulation, the effect on action potential duration develops at a progressively increasing rate and eventually gives rise to spontaneous depolariza- Cardioactive Substances that Prolong the Open State of Sodium Channels 39 tions arising from the prolonged repolarization phase. For a given effect, measured in terms of the prolongation of the action potential or the first appearance of a coupled extrasystole, the product of batrachotoxin concentration and number of preceding action potentials during exposure to that concentration of batrachotoxin is virtually constant. The effect thus fits the quantitative requirements of an irreversible one-to-one binding reaction of batrachotoxin molecules with sarcolemmal receptors that are accessible only during the action potential. As in the frog node of Ranvier (Sect. 3.3), the binding of batrachotoxin seems to require specifically the opening of Na channels. Prolonged depolarization by exposure to a high-K solution does not lead to the expression of the batrachotoxin effect in guinea-pig papillary muscle. Removal of batrachotoxin from the bathing solution during continued stimulation of the muscle causes the effect to decrease gradually over several hours, indicating a very slow dissociation of batrachotoxin. The dissociation of batrachotoxin (as manifested by the disappearance of its effect on the repolarization phase) does not require activity and can thus be demonstrated, even in the presence of toxin, simply by resting the muscle. Concomitantly with the prolongation of the action potential, batrachotoxin produces a positive klinotropic and positive inotropic effect and prolongs relaxation time in guinea-pig papillary muscle (Honer]iiger and Reiter 1977b). These effects persist in preparations from reserpine-pretreated animals, and are prevented by the presence of TTX (I0 #tool/ litre). The sensitivity to TTX suggests that the positive inotropic effect results from the increased Na influx induced by batrachotoxin. A reserpine-resistant, TTX-sensitive positive inotropic effect of batrachotoxin is also observed in the isolated cat papillary muscle (Shotzberger et al. 1976). In guinea-pig papillary muscle (Honer]iiger and Reiter 1977b), the rested-state contraction elicited during incubation of the resting muscle with high concentrations of batrachotoxin (0.6-1.2/~mol/litre) is increased in amplitude and rate of rise. This inotropic effect is absent in muscles from reserpine-pretreated guinea-pigs, pointing to the involvement of endogenous noradrenaline as in the case of high concentrations of veratridine or cevadine (Sect. 4.2.5).

4.4 Aconitine

4.4.1 Sinoatrial Node and Atrium In a preparation consisting of the sinoatrial node and extranodal tissue of the rabbit heart, aconitine (1 ug/ml) produces a bigeminal rythm, followed by flutter and irregular activity (Matsumura and Takaori 1959b). 40 P. Honerj~iger

The intracellular records demonstrate that both nodal and extranodal fibres participate in the abnormal rhythm.

4.4.2 Purkinje Fibres Aconitine produces very similar effects on Purkinje fibres isolated from dog (Schmidt 1960), calf or sheep hearts (Heistracher and Pillat 1962). Aconitine increases the rate of spontaneous depolarization thus causing a positive chronotropic effect on isolated Purkinje fibres. At this stage of action, parameters of the transmembrane action potential are not altered. Subsequently, aconitine selectively slows the terminal part of repolarization (at about -60 mV) which results in the appearance of after-depolarization. The duration of the after-depolarization increases from beat to beat until premature action potentials arise from the prolonged repolarization phase. Following a stage of bigeminy and trigeminy, a flutter-like activity results and, finally, the preparations are arrested at a reduced membrane potential (-55 to -45 mV). Decreasing the extracellular Na concentration to 10% of the normal level causes an immediate, though transient, interruption of the aconitine-induced spontaneous activity (Schmidt 1960). Peper and Trautwein (1967) analysed the effect of aconitine on sheep Purkinje fibres with a voltage-clamp technique. In the presence of aconitine (1 ug/ml) the current-voltage relation, obtained by changing the membrane potential at constant and relatively slow rates, shows an abnormal inward current with a maximum near - 60 inV. Application of TTX (63 umol/litre) blocks this current as well as the normal excitatory Na current. In Na-free solution, aconitine fails to affect the current-voltage relation. These findings suggest that aconitine abolished the inactivation gate in a fraction of Na channels, thus causing a persistently activated component of PNa"

4.3.3 Ventricular Muscle Matsuda et al. (1959) describe the effects of amorphous aconitine (Merck) and of the purified alkaloids aconitine and mesaconitine on isolated right ventricular tissue of the dog heart. No essential difference was noted among the three alkaloidal preparations. A few minutes after drug administration (0.01-0.1 ug/ml), the muscle suddenly undergoes a flutter- like, automatic and rapid excitation. Non-stimulated, resting preparations do not become spontaneously active if aconitine is added, and aconitine does not affect the resting transmembrane potential in these fibres. The automatic activity is shown to result from one of two mechanisms. Most frequently, the first visible effect is a decrease of the resting potential, followed by oscillatory potential changes following each action potential Cardioactive Substances that Prolong the Open State of Sodium Channels 41 and superimposed on the decreased level of resting potential. Schrnidt (1960), in his study on dog ventricular trabeculae, reported no oscillatory after-potentials under the influence of aconitine, but only a slowing of the final repolarization phase, which then led to premature action potentials. This mechanism of arrhythmogenesis was observed less frequently by Matsuda et al. (1959). Heistracher and Pillar (1962) report the effects of aconitine (0.05- 1.0 gg/ml) on transmembrane potentials of the isolated cat papillary muscle. The initial electrophysiologic effect appears to be a prolongation of the repolarization phase near its end (after-depolarization). The slowing ofrepolarization is less abrupt and less pronounced than that observed in dog ventricular muscle (Schmidt 1960). The after-depolarization may escape detection if the arrhythmogenic effect of aconitine develops rapidly. In the second stage of aconitine action, extrasystoles appear in the form of bigeminy or trigeminy. The premature action potentials seem to arise from the aconitine-induced after-depolarization. Subsequently, the papillary muscles show sustained high-frequency activity. The phase of automatic activity is followed by the termination of activity owing to a reduction of the resting membrane potential to about -55 mV in right ventricular muscle isolated from the rabbit heart (Matsumura and Takaori 1959a). An as yet unexplained effect of aconitine is the appearance of spike-like depolarizations arising from the normal resting potential and having an amplitude of up to 40 mV (Heistracher and Pillat 1962). In the isolated cat papillary muscle, the time to development of aconitine-induced automaticity is inversely related to aconitine concentration (0.25-1 ~g/ml), diastolic tension (60%-140% or peak tension) and stimulation frequency (1-4 Hz) during the preautomatic period (Tanz et al. 1973). As also noted by earlier investigators, the aconitine arrhythmia is not reversed by repeated washing of the preparations. The cumula- tive effect of repetitive stimulation may indicate that myocardial Na channels, like neural Na channels (Mozhayeva et al. 1977), interact with aconitine only when they are in the open configuration. The aconitine- induced arrhythmia israpidly abolished by 3/~mol/litre TTX (Tanz 1974). Although aconitine clearly induces a long-lasting activation of Na channels in Purkinje fibres (Peper and Trautwein 1967) and probably also in ventricular muscle, there is apparently no evidence that aconitine produces a positive inotropic effect. In this respect, aconitine differs from all other cardioactive Na channel-gate toxins reviewed in this chapter. It may seem questionable therefore whether an increase in sarcolemmal Na t~ermeability is strictly linked to positive inotropic action. One possible explanation for the failure of aconitine to produce a positive inotropic effect under the conditions used by previous investigators (e.g., Fig. 1 in Tanz et al. 1973) is that the transition to the state ofextrasystoles 42 P. Honerj~iger and spontaneous activity occurs without a sufficiently long-lasting phase during which aconitine increases the Na influx associated with each ~iction potential, but does not yet produce extrasystoles. In fact, while the stage of action characterized by the prolonged repolarization phase is easily demonstrated with many other Na channel-gate toxins, this has proven difficult with aconitine (Heistracher and Pillat 1962). A careful analysis o1~ the electrophysiologic and inotropic effect of aconitine in the prearrhythmic phase of action is clearly necessary to settle this point. We have recently observed that the arrhythmogenic effect of aconitine (0.5-1.0 ~mol/litre) is preceded by a small, but distinct positive klinotropic and inotropic effect in guinea-pig papillary muscle (P. Honerj@er and A. Meissner, unpublished work).

4.5 Grayanotoxins

4.5.1 Sinoatrial Node In the isolated sinoatrial node preparation of the rabbit heart, grayanotoxin I (0.1 mmol/litre) decreases the maximum diastolic potential, increases the rate of spontaneous diastolic depolarization, thereby causing an increase in firing frequency and, finally, inhibits the action potentials (Seyama 1978). These effects are fully reversible by washing with toxin-free medium. TTX (10 umol/litre) abolishes all effects on the transmembrane potentials, indicating that grayanotoxin I activates Na channels in the sinoatrial node. Thus, although this cell type is characterized by TTX-resistant electrical activity, the presence of Na channels can be demonstrated by pharmacological (Kreitner 1975) or electrical hyperpolarization (Noma et al. 1977) or by applying the Na channel-gate toxin grayanotoxin I (Seyama 1978).

4.5.2 Atrium In resting right atrial strips obtained from the rabbit heart, grayanotoxin I (0.1 mmot/litre) produces a marked depolarization (by 26 mV), which is reversed by the withdrawal of external Na ions or the application of 10 tamol/litre TTX (Seyama 1978). A less-pronounced depolarization is observed with lower concentrations of grayanotoxin I or a-dihydro-grayanotoxin II applied to isolated and stimulated left atria of the guinea-pig (Akera et al. 1976; Ku et al. 1977). The addition of TTX (0.8 umol/litre) reverses the depolarization produced by grayanotoxin I (0.5/amol/litre) and partially restores the amplitude of the action potential, which is reduced by grayanotoxin I owing to the reduced resting potential (A kera et al. 1976). Thus grayanotoxin I presumably induces a persistent activa- Cardioactive Substances that Prolong the Open State of Sodium Channels 43 tion of Na channels resulting in depolarization of the resting membrane. Concentrations of grayanotoxin I that alter themembrane potential also cause a positive klinotropic and positive inotropic effect under isometric conditions (Akera et al. 1976). This effect is inhibited by TTX (0.8/amol/ litre). The racemic mixture of propranolol (10 ~mol/litre) also inhibits the positive inotropic effect of both grayanotoxin I and a-dihydro-grayanotoxin II (Ku et al. 1977). These authors state "that a portion of the effects of grayanotoxins on isometric contractile force may be mediated by a beta-adrenergic mechanism". However, the Na channel blocking action of propranolol (Morales-Aguillera and Vaughan Williams 1965) might be responsible for this antagonism. At 5 ~mol/litre, grayanotoxin I produces arrhythmia in isolated guinea-pig left atria (Akera et al. 1976). The grayanotoxins enhance the ouabain-sensitive uptake of the potassium analog rubidium by guinea-pig ventricular slices, which is interpreted as a stimulation of the Na-K pump secondary to the toxin-induced increase of Na influx (Ku et al. 1977). The grayanotoxins do not affect Na, K- ATPase isolated from rat brain or guinea-pig heart (Akera et al. 1976; Ku et al. 1977).

4.5.3 Atrioventricular Node There are no reports on the effect of grayanotoxins on the transmembrane electrical activity of cells of the atrioventricular node. In the dog heart-lung preparation, examined with extracellular electrodes, grayanotoxin I (andromedotoxin) prolongs the atrioventricular conduction time and increases the spontaneous firing rate of the atrioventricular node if the faster rhythm of the sinoatrial node is suppressed (Swain and McCarthy 1957).

4. 5. 4 Purkin/e Fibres

Conduction through the Purkinje system is reported to be slowed by grayanotoxin I (andromedotoxin) in the dog heart (Swain and McCarthy 1957). This occurs at a time when ventricular myocardial conduction is not affected. Possibly, the effect on Purkinje fibre conduction is related to a depolarization as observed in atrila cells (Sect. 4.5.2).

4. 5. 5 Ven tricular Muscle Hotta et al. (1980) studied 18 structurally related grayanotoxins which produced a concentration-dependent reversible positive inotropic effect on isolated guinea-pig papillary muscle. 44 P. Honerjgger

4.6 Scorpion Toxins

4. 6.1 Isolated Mammalian Heart

Corrado et al. (1968) investigated the effects of crude dried venom of the Brazilian scorpion Tityus serruluatus on the isolated guinea-pig heart. In final concentrations of approximately 1 gg/ml, the venom produces a short-lasting bradycardia followed by an increase of force and frequency of contractions. The effects are reversible and reproducible in the same preparation. The bradycardia is blocked by atropine and potentiated by neostigmine. The positive chronotropic and inotrpic action is abolished by propranolol or bretylium. It is absent in hearts of reserpine-pretreated guinea pigs. Hexamethonium neither affects the bradycardia nor the tachycardia in doses which abolish the nicotine-induced bradycardia and positive inotropic effect. Hence the venom probably acts on the postganglionic nerve terminals to cause the release of acetylcholine and noradrenaline. Langer et al. (1975) have shown that a purified toxin from T. serrulatus increases the release of 3 H-noradrenaline from guinea-pig atria. Less extensive experiments were performed by other investigators with venoms of Leiurus quinquestriatus, Buthus minax, B. occitanus, and Androctonus australis HECTOR using the isolated heart of rabbits or guinea pigs. Similar to Tityus scorpion venom, the other scropion venoms seem to alter cardiac function mainly through the release of neurotransmitters from intracardiac nerve fibres (Zlotkin et al. 1978). Coraboeuf et al. (1975) used the isolated rat heart to study the effects of pure ScTXIt on contraction and transmembrane electrical activity (at 24°-25°C). ScTXII produces a reserpine-resistant positive inotropic effect that is associated with an increase in the height of the plateau of the ventricular action potential and a prolongation of the repolarization phase. The effect on the action potential persists for hours following a single injection of toxin into the coronary arteries. Resting potential and Vmax are only slightly altered. The lengthening of the action potential produced by ScTXII is greatly diminished by injections of TTX, Ca- rich medium or procaine, and it is prevented during perfusion with low-Na solution. The atrial action potential is also prolonged by ScTXII. According to Coraboeuf et al. (1975), ScTX n increases plateau height by slowing down the inactivation of Na channels or by inducing an incomplete Na inactivation. The effect on the action potential might indirectly favour the penetration of Ca through the slow channel because of a stronger and longer-lasting activation of the potential- and time- dependent slow inward Ca current, and this could explain the positive inotropic effect. The authors do not discuss the possibility of increased Na-Ca exchange. Cardioactive Substances that Prolong the Open State of Sodium Channels 45

Confirming earlier investigations, Coraboeuf et al. (1975) find that ScTXII produces a positive inotropic effect also on isolated guinea-pig and rabbit hearts, but that this effect is absent in preparations obtained from reserpine-pretreated animals. Guinea-pig and rabbit ventricular action potentials are insensitive to the scropion toxin in doses that are markedly effective in the rat heart. As pointed out by Coraboeuf et al. (1975), this suggests the existence of species differences in the sensitivity of myocardial Na channels to scorpion toxin.

4.6.2 Cardiac Cells in Culture

ScTXII has marked effects on the mechanical and electrical activity of nerve-free myoblast cultures derived from the chick embryonic heart (Fayet et al. 1974; Bernard and Couraud 1979). ScTXII increases the frequency of the spontaneous contractions, and this effect is followed by cell fibrillation and contracture (Fayet et al. 1974). The bioelectric cause of these phenomena was elucidated by Bernard and Couraud (1979). They used cells from 11-day-old chick embryonic hearts cultured as aggregates in suspension. With their culture technique, they obtain two populations of cells which differ in their value of Vmax ("fast" and "slow" cells). The effect of ScTXII depends on the type of cell.

Fast Cells. This population of cells has resting potentials between -40 and -72 mV. Vmax exceeds 15 V/s and is significantly related to the level of the resting potential, reaching a value of 125 V/s at the most negative resting potential. TTX (0.4 #mol/litre) reduces Vmax by about 90%. ScTXII (30 nmol/litre) causes approximately a tenfold increase in the duration of the action potential, while it does not significantly alter Vmax. TTX (0.1 t~mol/litre) rapidly suppresses the toxin-induced modification of the action potential. D 600, the methoxy derivative of verapamil, at a concentration of 1 tsmol/litre, does not prevent prolongation of the action potential by ScTXII. These results suggest that ScTXtI prolongs the open state of Na channels in cultured cells with expressed Na channels, perhaps by slowing inactivation as in neural Na channels (Sect. 3.6).

Slow Cells. This population of cells has resting potentials between -40 and - 62 mV, and Vmax is 10 V/s or less, irrespective of the resting potential, and unaffected by TTX at 0.4 pmol/litre. The low value of Vmax, its lack of correlation with the level of the resting potential, and its insensi- tivity to TTX indicate that the upstroke of the action potential is not mediated by fast Na channels. In these cells, ScTXII (30 nmol/litre), in addition to prolonging the repolarization phase of the action potential, increases Vmax significantly from 8.6 to 36.8 V/s on average, while having 46 P. Honerj~iger no effect on the level of the resting membrane potential. Both effects are suppressed by TTX (0.1 #mol/litre). ScTXII restores action potentials in cells when electrical activity was previously reduced by D 600 (0.5 #mol/ litre). Bernard and Couraud (1979) conclude that ScTXII increases Vmax (in slow cells) and prolongs the repolarization phase of the action potential (in both types of cells) by retarding the inactivation of Na channels. The fact that ScTXII conveys TTX sensitivity to the electrical activity of slow cells shows that these normally TTX-resistant cells contain silent Na channels (Sect. 3.9).

Effect on Na and Ca Influx. ScTXII causes a TTX-sensitive increase of Na influx in embryonic cardiac cells (Couraud et al. 1976), as might be expected from the electrophysiological actions described above. The influx of Ca ions is enhanced as well. The effect of Na channel-gate toxins on Ca influx in cultured heart cells is discussed in the section on Na-Ca exchange (Sect. 5.3.2).

4.7 Sea Anemone Toxins

4. 7.1 Toxin H from Anemonia sulcata

Isolated Mammalian Heart. In the heart-lung preparation of the cat, ATXII produces a positive inotropic effect (Alsen et al. 1976). The threshold concentration is approximately 2 nmol/litre, and a maximum positive inotropic effect occurs at around 100 nmol/litre. In the inotropic concentration range, ATXII has no significant chronotropic action. Toxic symptoms occur at 160 nmol/litre. They are characterized by periods ofventricular fibrillation alternating with periods of normal spontaneous rhythm. In the isolated guinea-pig heart stimulated at 3 Hz, ATXII produces a half-maximal positive inotropic effect at about 5 nmol/litre on the ventricle and atrium (Alsen et al. 1976). Arrhythmia occurs at 25 nmol/litre ATXII.

Atrium. In guinea-pig atrium, ATXII (10 nmol/litre) prolongs the repolarization phase of the action potential at all levels of membrane potential and produces a positive klinotropic and positive inotropic effect (Ravens 1976). The prolonged phase of repolarization is associated with a prolongation of the relaxation phase of the isometric contraction.

Ventricular Muscle. Ravens (1976) analysed the effects of ATXII on the transmembrane electrical activity and contraction of the isolated guinea- pig papillary muscle contracting at 1 Hz. At concentrations of 5-20 nmol/ Cardioactive Substances that Prolong the Open State of Sodium Channels 47 litre, the toxin produces graded positive klinotropic and positive inotropic effects. They reach steady-state in about 30 rain and disappear within about 1 h of superfusion with toxin-free solution. Inotropic concentrations induce a prolongation of the action potential at negative levels of membrane potential. This prolongation is only partially reversed during a 1-h washout period. Concentrations up to 20 nmol/litre leave the resting membrane potential and the amplitude of the action potential unaffected. Vmax is slightly reduced by 20 nmol/litre ATXII. The prolonged repolarization phase is associated with a prolongation of the relaxation phase of the isometric contraction. The effects of ATXII are not modified by catecholamine depletion with reserpine. TTX (0.5 t~mol/litre) completely and reversibly inhibits the effect on action potential duration and the positive inotropic effect. ATXII (10 nmol/litre) fails to prolong the action potential and to increase force of contraction if extracellular K is raised from 2.7 to 14.7 mmol/litre, and raising the K concentration after the effects of ATXI! have developed causes their disappearance. The sensitivity to TTX of the toxin-modified repolarization phase suggests that it is due to the persistent activation of Na channels. This might result from a delayed inactivation of the Na channels, which is the main effect of ATXII on neural Na channels (Sect. 3.7.1).

Cardiac Cells in Culture. Romey et al. (1980) analysed the effects of ATXII on cardiac cell aggregates in culture obtained from 3- and 1 &day- old chick embryo hearts, respectively. The younger cells exhibit low values of Vmax and their action potential is insensitive to TTX; they will be referred to as slow cells. The older cells show values of Vmax greater than 100 V/s and their action potentials are blocked by TTX (0.1 #tool/ litre); these cells will be called fast cells. Very similar to scorpion toxin (Sect. 4.6.2), ATXI1 produces different effects on slow and fast cells. In fast cells, ATXII only prolongs the falling phase of the action potential and thus produces qualitatively the same effect as observed in adult guinea-pig myocardial cells (Ravens 1976). In slow cells, ATX n produces an increase of Vmax (from 12.5 to 25 V/s on average), in addition to prolonging the falling phase of the action potential. The increase of Vmax occurs under conditions where any steady-state inactivation of the Na channels is prevented by a long-lasting hyperpolarizing prepulse. In fast cells, TTX (0.1 umol/litre) slows Vmax and abolishes the effect of ATXII on the repolarization phase. The same concentration of TTX blocks the two effects of ATXII on the action pontential of slow cells, namely the increase in Vmax and the prolongation of the falling phase. These results as well as those obtained with scorpion toxin (Sect. 4.6.2) suggest that the polypeptide toxins slow Na inactivation in fast cells, thereby prolonging the repolarization phase of the action potential. In slow cells, 48 P. Honerj~iger the polypeptide toxins cause the appearance of otherwise silent Na channels, as suggested by the TTX-sensitive increase in Vrnax , and these Na channels seem to inactivate only slowly, as shown by the TTX-sensitive prolongation of the repolarization phase. Thus, ATXII reveals Na channels which are not normally expressed in embryonic heart cells derived at an early stage of development. In addition, Romey et al. (1980) showed that ATXII exerts a positive inotropic effect on embryonic cardiac cells along with its effect on the action potential. The inotropic effect is blocked by TTX (0.1 pmol/litre) suggesting that it is causally related to the modification of the Na channels by ATXII. The link may be increased Na-Ca exchange across the sarcolemma, as indicated by the observation that ATXII increases Ca influx as well as Na influx (Romey et al. 1980). The effect of Na channel-gate toxins on Ca influx is discussed in the section on Na-Ca exchange (Sect. 5.3.2).

4. Z2 Anthopleurin A

Sinoatrial Node. At concentrations of 5 or 30 nmol/litre, anthopleurin A has no significant effect on the spontaneous heart rate of isolated atria from the rabbit, cat, guinea pig or rat (Shibata et al. 1976).

Atrium. Anthopleurin A produces a reversible positive inotropic effect on isolated left atria from the rabbit, cat, guinea pig or rat at a contraction frequency of 1.6 Hz (Shibata et al. 1976). Half-maximally effective concentrations are 1-3 nmol/litre. The effect is not modified by propranolol (1 umol/litre). TTX (2 umol/litre) decreases the sensitivity of guinea-pig atria to the positive inotropic action of anthopleurin A (Shibata et al. 1978). Anthopleurin A (50 nmol/litre) does not alter the cAMP content of guinea-pig atria (Shibata et al. 1976).

Purkin]e Fibres. Shimizu et al. (1979) studied the effects of anthopleurin A on the transmembrane electrical activity of isolated canine Purkinje fibres and ventricular muscle fibres in a preparation containing fibres of the right bundle branch and of the anterior papillary muscle. In Purkinje fibres stimulated at 1.67 Hz, anthopleurin A (3.9-39 nmol/litre) prolongs the repolarization phase of the action potential by prolonging the duration of the plateau phase. It has little effect on the subsequent phase of repolarization, i.e. the times to 50% and 90% repolarization are prolonged to an equal extent. The refractory period is increased along with the prolongation of the plateau phase of the action potential. At high concentrations of the polypeptide (19-39 nmol/litre), premature action potentials develop from the prolonged repolarization phase at potential levels between -10 and -50 mV. Up to the highest concentration tested Cardioactive Substances that Prolong the Open State of'Sodium Channels 49

(39 nmol/litre), anthopleurin A does not significantly affect the resting membrane potential, amplitude or Vmax of the action potential. In concentrations below 19 nmol/litre, anthopleurin A produces no significant change in the firing rate of spontaneously active Purkinje fibres. Shimizu et al. (1979) did not test whether TTX inhibits the effect of anthopleurin A on the repolarization phase, as has been observed in the presence of batrachotoxin (Hogan and Albuquerque 1971) or veratrine (Arbel et al. 1975). An inhibition by TTX would be expected if anthopleurin A slows the inactivation of Purkinje fibre Na channels as it does that of neural Na channels (Sect. 3.7.2) and ventricular myocardial Na channels.

Ventricular Muscle. Anthopleurin A increases the force of contraction of isolated papillary muscles and ventricular strips of the guinea-pig heart driven at 1.6 Hz (Shibata et al. 1976). Half-maximally effective concentrations are 4.4-4.8 nmol/litre. At a threshold concentration near 2 nmol/ litre, anthopleurin A also increases the force of the isolated cat papillary muscle stimulated at 1 Hz (Scriabine et al. 1979): As shown in this study the positive inotropic and positive klinotropic effects of anthopleurin A are associated with a marked prolongation of the relaxation phase (at 64 nmol/litre). Arrhythmia is produced by 120 nmol/litre. As in dog Purkinje fibres, but to a smaller extent, anthopleurin A increases the action potential duration and the refractory period in dog ventricular muscle fibres (Shimizu et al. 1979): The prolongation of the repolarization phase is already manifest at positive levels of membrane potential. In this tissue, anthopleurin A, at concentrations of up to 39 nmol/litre, does not affect the resting potential, Vmax, or the amplitude of the action potential. Using a voltage-clamp technique (single sucrose gap) on the isolated guinea-pig papillary muscle, Hashimoto et al. (1980) have recently shown that anthopleurin A (9.7-39 nmol/litre) induces a maintained inward current during depolarizing pulses superimposed on the outward current and reaching a maximum value between -40 and -20 inV. The inward current represents a persistent component of Na flux through Na channels since it is blocked by TTX. TTX likewise abolishes the prolongation of the action potential produced by anthopleurin A in guinea-pig papillary muscle (Hashimoto et al. 1980). Ochi and colleagues (Hashirnoto et al. 1980) were the first to suggest that anthopleurin A produces its positive inotropic effect by increasing Na influx. Anthopleurin A (50 nmol/litre) does not affect the activity of Na, K-ATPase isolated from guinea-pig or dog hearts, of phosphodiesterase isolated from guinea-pig hearts, and it does not alter the cAMP content of guinea-pig ventricle (Shibata et al. 1976). 50 P. Honerjgger

4.8 Coral Toxin

Tbe cardiac action of goniopora toxin was examined in isolated left atrial preparations of the rabbit (Fu]iwara et al. 1979). At 30 nmol/litre, the polypeptide toxin prolongs the action potential at all levels of repolarization, while no significant change is observed either in the resting membrane potential, the overshoot or Vmax" The effect on action potential duration persists during superfusion with toxin-free solution for 2 h. TTX (1 #mol/litre) abolishes the effect on the replarization phase. At 3-100 nmol/litre goniopora toxin produces a positive inotropic effect that also persists during washout with toxin-free solution. In line with an irreversible binding reaction, the positive inotropic effect during exposure to a given concentration of the toxin does not reach a steady-state, but progresses and is eventually followed by arrhythmia characterized by coupled extrasystoles. Increasing the concentration of the toxin acceler- ates the development of the positive inotropic effect. Propranolol, phen- ~tolamine or atropine (each at 1 /~mol/litre) do not modify the positive inotropic effect. The positive inotropic and arrhythmogenic effect are both abolished by TTX (1/~mol/litre). In atria depolarized by an elevated extracellular K concentration (27 mmol/litre) and treated with 0.5/lmol/ litre isoprenaline to restore excitability (0.25 Hz), goniopora toxin fails to increase force of contraction. The results suggest that goniopora toxin modifies the cardiac action potential by prolonging the open state of Na channels and that the positive inotropic effect is linked to the increase in sarcolemmal PNa"

5 Sarcolemmal Na Permeability and Myocardial Force of Contraction: Na-Ca Exchange

5.1 Introduction

For an understanding of the positive inotropic action of the Na channel- gate toxins it is necessary to consider the role played normally by Na channels in the excitation-contraction cycle of the heart. The most important function of the Na channels is to allow a rapid conduction of the excitation wave, originating in the sinoatrial or atrioventricular node, to all cells of the atrial and ventricular myocardium. The nearly simultaneous excitation of all atrial and ventricular cells assures the coordinated contraction of these cells, i.e. the function of the heart as a pump. The process which couples sarcolemmal excitation to contraction of the myocardial cell does not seem to depend so much on the activation of Na Cardioactive Substances that Prolong the Open State of Sodium Channels 51 channels, but seems to be intimately related to events occurring during the plateau phase of the action potential, notably the Ca inward current (Beeler and Reuter 1970a,b,c; New and Trautwein 1972; Trautwein et al. 1975) and electrogenic Na-Ca exchange. Thus, contractions can still be elicited if Na channels are blocked by TTX or completely inactivated by sustained depolarization of the sarcolemma. It is nevertheless very likely that Na channels, in addition to permitting the rapid conduction of the cardiac action potential, influence excitation-contraction coupling, because their activation leads to an influx of Na ions into the cell. Several lines of evidence, reviewed here, suggest that an increased concentration of intracellular Na ions promotes an influx of Ca ions across the sarcolemma (Na-Ca exchange). This is expected to result in a positive inotropic effect. Na influx through Na channels is greatly enhanced by the Na channel-gate toxins, because they drastically prolong the time during which the Na channels are in the open state. The intracellular Na concentration may also affect the Ca transport by organelles of the myocardial cell. If exposed to Na ions, cardiac mito- chondria (Carafoli and Crornpton 1978) release Ca. It seems difficult, however, to explain a maintained positive inotropic effect by a mechanism that depletes cellular stores and thus, eventually, the cell of Ca ions. A decrease of the [K]/[Na] ratio may impair Ca uptake by cardiac sarcoplasmic reticulum (Katz and Repke 1967). Ravens (1976) suggested an explanation for the positive inotropic effect of Na channel-gate toxins that does not involve changes of intracellular Na concentration. According to this hypothesis, the toxin inter- acts with a sarcolemmal receptor controlling Na channel gating as well as a plasmalemmal Ca store that releases Ca during contraction. However, since TTX, a selective blocker of Na channels, abolishes the postive inotropic effect of Na channel-gate toxins (see Chap. 4), it is not necessary to postulate that the positive inotropic effect of these toxins results from a modification of structures other than those regulating Na channel gating.

5.2 Na-Ca Exchange in the Squid Giant Axon

The giant axons of certain species of squid are particularly suitable for studying the role of intracellular Na in regulating Ca flux across an excitable membrane, because [Na]i can be selectively altered experimentally either by injection of a Na salt into the axoplasm of an intact axon (Baker et al. 1969) or by changing the concentration of Na in the solution used to perfuse an axon intracellularly (Di Polo 1979). Baker et al. (1969) showed that the intraaxonal injection of NaC1 to raise [Na]i by 67 or 120 mmol/litre in axons of Loligo forbesi previously depleted of 52 P. Honerj~iger intracellular Na causes an increase in the unidirectional influx of Ca ions by factors of 2 and 4, respectively. These experiments were performed with an external solution in which most of the Na was replaced by Li ions (Li-seawater). In other experiments of their study [Na] i was raised by prolonged stimulation at high frequency. This resulted in an increase of Ca influx irrespective of whether the influx was measured in Na-, Li- or dextrose-seawater. The additional demonstration of a component of Na efflux, which is insensitive to cardioactive steroids, but activated by external Ca ions, led to the hypothesis that the enhancement of Ca influx by internal Na involves a transmembrane ion exchange in which the exit of Na ions is linked to the entry of Ca ions (Baker et al. 1969). A detailed analysis of the component of Ca influx enhanced by intracellular Na ions was performed by Di Polo (1979) using the technique of intracellular dialysis on axons of the tropical squid Dorytheutis plei. The main findings of this study may be summarized as follows: An increase of [Na] i of 0-120 mmol/litre increases Ca influx, and the Na i- dependent Ca influx requires a threshold level of [Ca2+]i of 40 nmol/litre and increases as [Ca2+]i is raised to 0.8 t~mol/litre. The Nai-dependent Ca influx requires the presence of ATP. The results of Baker et al. (1969) indicate that the entry of one Ca ion may be linked to the exit of more than two Na ions, which would make Na-Ca exchange sensitive to the membrane potential. In line with this prediction, both the Na efflux component (Baker and McNaughton 1976) and the Ca influx component (Di Polo 1979) constituting the Na-Ca exchange under physiological conditions, i.e. in the presence of ATP, were found to be augmented by depolarization. Baker et al. (1969) pointed out that the exchange of external Ca for internal Na which they observed may represent ion movements in a direction opposite to that occurring under normal conditions. The normal function of a Na-Ca exchange system might be to pump Ca out of the cell in exchange for external Na, as suggested by Reuter and Seitz (1968). In this case the energy for the uphill transport of Ca ions from the cell could be provided by the downhill movement of Na ions, and the transmembrane gradient for Na ions would be maintained by the Na pump. Interestingly, although a Ca efflux-Na influx system operates in axons poisoned with cyanide (Blaustein and Hodgl~n 1969), later work has shown that 50%-90% of the Ca efflux from unpoisoned squid axons is independent of external Na ions and requires the presence of ATP (Baker and McNaugh ton 1978; Di Polo and Beaug~ 1979). The effect of intracellular Na ions on unidirectional Ca efflux has also been examined. Raising [Na]i from 5 to 75 mmol/litre in internally dialyzed axons of Loligo pealei inhibits the [Na]o-dependent component of Ca efflux (Blaustein 1977), but the uncoupled Ca efflux, which may Cardioactive Substances that Prolong the Open State of Sodium Channels 53 constitute the main part of Ca efflux from intact axons (Baker and McNaughton 1978), is insenstitive to changes in intracellular Na concentration (Di Polo and Beaugd 1979). Thus, an increase of [Na]i will affect the intracellular Ca concentration of squid axon through an increase of Ca influx with little effect on Ca efflux.

5.3 Nai-Ca o Exchange in Cardiac Preparations

5. 3.1 Guinea-PigAtrium Glitsch et al. (1970) examined the relationship between the intracellular Na concentration and Ca influx in resting left atria of guinea pigs. They produced different levels of [Na]i by equilibrating the resting muscles in K-rich solution ([Na]i = 12.5 mmol/kg fibre water, as determined by flame photometry), in normal solution ([Na]i = 20 mmol/kg)or in cold K-poor, Ca-poor solution ([Na]i = 60 mmol/kg). An intermediate level of Na i was established by high-frequency stimulation in K-poor, Ca-poor solution ([Na]i = 40.5 mmol/kg). Subsequently all preparations were placed for 10 min into a medium containing 4s Ca, 0.65 mmol/litre [K]o to prevent a decrease of [Na]i , and 0.7-7 ~mol/litre acetylcholine to prevent spontaneous activity of the atria. After the uptake period the preparations were washed for 25 min with inactive solution before the radioactive analysis was performed. Under these conditions, the increase of [Nali was associated with an increase of Ca uptake by the atria. A nearly fivefold increase in [Na]i caused in increase in Ca uptake by a factor of about 2.6.

5.3.2 Cardiac Cells in Culture To define the relationship between Na fluxes and Ca fluxes in myocardial cells an approach different from that of Glitsch et al. (1970) was used by Fosset et al. (1977). They analysed the Ca influx induced by veratridine in monolayer cultures of cardiac myoblasts obtained from 10-day-old chick embryos. Although these authors did not report the actual changes of [Nali in their experiments, the various concentrations of veratridine which they applied in a medium containing Na ions (140 mmol/litre) and ouabain increased Na influx and presumably caused a graded increase of [Na]i. No information on the transmembrane potential of the myoblasts is given in this article, but the work by Sperelakis and Lehmkuhl (1968) and Sperelakis and Pappano (1969) suggests that either ouabain or the lowest effective concentration of veratridine (1.5 #mol/litre) used by Fosset et al. (1977) caused permanent depolarization and inexcitability and thus eliminated possible complications resulting from spontaneous 54 P. Honerj~iger

25 Fig. 12. Concentration-effect relationships for the enhancement of Na influx and Ca influx by veratridine in 80 20 chick embryonic cardiac cells in culture. The experiments were performed .70 "6 in the presence of 0.5 mmol/litre ouabain. (Drawn after Fosset et al. ~'60 .c_ c 1977) £ )so 10-6 ~40 Na influx A

2o3o*~o~ - Ca influx 10 100 ~mot/t Veratridine concentration activity of cultured heart cells. Fosset et al. (1977) showed that veratridine increases Ca influx up to 25-fold (Fig. 12). They could exclude the possibility that this influx uses Na channels opened by veratridine, because veratridine fails to increase Ca influx if external Na is replaced by Li ions, i.e. under conditions where veratridine retains the ability to open Na channels thereby allowing the influx of Li ions (Sperelakis and Pappano 1969). However, because it is abolished by TTX, the veratridine effect on Ca influx does require the opening of Na channels. Veratridine- induced Ca influx is insensitive to manganese or cobalt ions or to the compound D 600 at concentrations at which these substances normally inhibit slow Ca channels. Thus, the veratridine-induced Ca influx is coupled to the presence of an inwardly directed Na gradient and open Na channels, but the Ca ions enter the cell neither through Na channels nor through slow Ca channels. These findings strongly suggest the existence in cardiac cells of a Ca uptake mechanism that is increased by intracellular Na accumulation (Fosset et al. 1977). Results very similar to those of Fosset et al. (1977) were obtained by Couraud et al. (1976) who used ScTXII. The latter authors suggest that the sarcolemma of cardiac cells in culture contains two types of Ca channels, the well-known slow Ca channel and a Ca channel that is "coupled to the passive Na action potential ionophore" and therefore affected by the toxin. This interpretation disregards the observation by the authors themselves that ouabain greatly enhances the effect of ScTXII on Ca influx, an observation which points to the importance of intracellular Na accumulation in linking the effect of the scorpion toxin on Na channels to its effect on Ca influx. It appears that all the results of Couraud et al. (1976) are compatible with the concept that ScTXII increases Na influx Cardioactive Substances that Prolong the Open State of Sodium Channels 55 by opening Na channels and thereby indirectly activates an exchange of intracellular Na for extracellular Ca ions. An increase of both Na and Ca influx is also produced by ATXII (Romey et al. 1980).

5. 3. 3 Purkinfe Fibres

The experiments of Glitsch et al. (1970), Couraud et al. (1976) and Fosset et al. (1977) do not clarify whether the activating effect of intracellular Na ions on Ca influx involves an actual exchange of internal Na ions for external Ca ions, as in the squid giant axon (Baker et al. 1969). If the cardiac sarcolemma possesses a Na-Ca exchange system, raising the extracellular Ca concentration will increase Na efflux thereby reducing the intracellular Na concentration under conditions where [Na]i is not controlled by the Na pump. Using Na-sensitive glass micro-electrodes, Deitmer and Ellis (1978) indeed showed that raising the bath concentration of Ca produces a decrease in the intracellular Na activity of sheep heart Purkinje fibres even when the Na-K pump is inhibited by strophan- tidin.

5.3.4 Myocardial Contraction in Low Extracellular Na

If extracellular Na is reduced in the presence of extracellular Ca, a contracture develops which is associated with a large increase in the influx of Ca into the heart cells (Niedergerke 1963). A study on frog atrial trabeculae revealed that this contracture is transient and, after spontaneous relaxation, even a 100-fold increase in the bathing Ca concentration (from 0.1 to 10 mmol/litre) fails to induce any increase in tension (Chap- man 1974). The results obtained by Chapman (1974, 1979) stongly suggest that the increase in Ca influx resulting from the reduction of [Na] o critically depends on the presence of Na inside the cells. This points to the existence of a Na-Ca exchange mechanism. Voltage-clamp experiments on frog atrial trabeculae (Benninger et al. 1976; Horackova and Vassort 1979a) indicate that the tonic component of tension (Sect. 6.1) results from a transmembrane Na-Ca exchange that is voltage-dependent and therefore activated by the depolarization used to elicit tonic tension. The tonic component of tension exists in addition to a phasic component that is related to both Is~ and intracellular Ca stores (Horackova and Vassort 1976, 1979b).

5. 3.5 Inotropic Effect of Cardioactive Steroids

If one accepts the view that cardioactive steroids produce their positive inotropic effect by inhibiting the sarcolemmal Na pump, one has to conclude that the myocardial cell possesses a mechanism that translates some 56 P. Honerj~iger consequence of this membrane effect into an enhancement of the intracellular Ca transient during contraction (Allen and Blinks 1978) and thus into a positive inotropic effect. One consequence of the Na pump inhibition by cardioactive steroids is an increase in intracellular Na concentration (Lee et al. 1980). A Na-Ca exchange in the sarcolemma would thus provide exactly the mechanism to couple an inhibition of the Na pump to a positive inotropic effect. A kera and Brody (1978) and R eiter (to be published) reviewed the evidence in support of the theory that the positive inotropic effect of cardioactive steroids results from their effect on [Na] i secondary to the inhibition of the Na pump.

5.3. 6 Cardiac Membrane Vesicles

A transmembrane Na-Ca ion exchange was demonstrated in membrane vesicles of presumed sarcolemmal origin. The vesicles were obtained by centrifugation techniques from the ventricle of the heart of the rabbit (Reeves and Sutko 1979), dog (Pitts 1979), and ox (Miyamoto and Racker 1980). Recent studies indicate that this Na-Ca exchange involves the exchange of more than two Na ions with one Ca ion and is thus electrogenic (Reeves and Sutko 1980; Philipson and Nishimoto 1980). Na channel-gate toxins have apparently not been used to alter Na fluxes of cardiac membrane vesicles. The demonstration of a toxin-activated and TTX-inhibited Na flux would provide strong evidence for the sarcolemmal nature of such vesicles. The need for further purification of cardiac sarcolemmal membranes has been stressed by Sulakhe and St. Louis (1980).

6 Comparison of Cardiac Actions of Na Channel-Gate Toxins with Those of Other Positive Inotropic Drugs

As shown in Chap. 4, the cardiac action of Na channel-gate toxins has been analysed and characterized in terms of their influence on mechanical and electrical activity and on transsarcolemmal ion movements. The purpose of the following paragraphs is to compare some of these effects with those of other classes of positive inotropic agents on the same aspects of myocardial function. This will serve to reinforce the conclusion that Na channel-gate toxins represent a separate class of positive inotropic substances characterized by a distinct mode of action. The discussion deals mainly with effects observed in isolated ventricular preparations of the mammalian heart. Cardioactive Substances that Prolong the Open State of Sodium Channels 57

6.1 Isometric Contraction Curve

Na channel-gate toxins increase the force of myocardial contraction by accelerating the rate of force development, i.e. by a positive klinotropic effect (Figs. 2, 3). Hence, they act by increasing the intensity of the "active state" of the contractile elements and differ in this respect from the relatively few interventions which, like the addition of fluoride, produce a positive inotropic effect by prolongation of the active state (Reiter 1972a). Na channel-gate toxins produce variable effects on the relaxation phase of the isometric contraction. The relaxation time may be prolonged to such an extent that total contraction time is lengthened (Fig. 2, veratridine), or the prolongation may just compensate a simultaneous abbreviation of time to peak force resulting in a nearly unchanged total contraction time (Fig. 3, germitrine). The very marked prolongation of relaxation time, causing an increase in total contraction time, is observed in the presence of those toxins which slow repolarization during, or shortly after, the plateau phase of the action potential (e.g., cevadine, veratfidine, batrachotoxin, ATXII , anthopleurin A, goniopora toxin; see Chap. 4 for references). In contrast, relatively little effect on the total contraction time is produced by those toxins inducing a slight depolarization of the resting membrane potential (e.g., germitrine, grayanotoxins). It is well known that an electrical prolongation of the repolarization phase (by injecting a depolarizing current with the sucrose-gap technique), at a level positive to about -30 mV, is associated with prolonged relaxation phase of the contraction (Morad and Trautwein 1968). The resulting component of mechanical activity is maintained as long as the membrane is kept at the depolarized level of membrane potential and is therefore called "tonic" tension (Morad and Goldman 1973). The markedly prolonged relaxation phase obse~wed with some of the Na channel-gate toxins can thus be explained in terms of the known dependence of mechanical activity on membrane potential. Recent studies indicate that Na channel- gate toxins may prolong relaxation time by an additional mechanism related to the increase in intracellular Na concentration rather than to the prolonged repolarization phase. Vassort and colleagues (Roulet et al. 1979) have shown that veratrine prolongs the relaxation time of frog atrial trabeculae even if the preparation is activated by voltage-clamp depolarizations of constant duration, i.e. if the prolongation of the action potential produced normally by veratrine is prevented. According to Roulet et al. (1979), the relaxation phase is directly influenced by an electrogenic sarcolemmal Na-Ca exchange mechanism. An increase in Na i is thus expected to decrease the efficiency of the Na-Ca exchange to restore the low diastolic level of the free intracellular Ca concentration 58 P. Honerj~iger after repolarization. This interpretation could also explain the prolongation of relaxation time produced by Na channel-gate toxins which, like germitfine (Fig. 3), prolong the action potential only at membrane potential levels far negative to -30 mV (Sect. 4.2.5). Na channel-gate toxins have never been observed to abbreviate total contraction time by shortening relaxation time. Such a relaxant effect is typically produced by inotropic agents which elevate the cellular concentration of cAMP, for instance by/3-adrenoceptor agonists, histamine, or phosphodiesterase inhibitors (Korth 1978; Scholz 1980). While the inotropic effect of the toxins clearly differs in this respect from the cAMP- increasing drugs, it is difficult, if not impossible, to distinguish certain other positive inotropic agents from Na channel-gate toxins solely on the basis of the modification of the isometric contraction curve. Thus, like germitrine, the cardioactive steroid dihydro-ouabain produces a positive klinotropic and inotropic effect with little effect on total contraction time (Reiter 1972b).

6.2 Blockade of Sarcolemmal Na Channels by Tetrodotoxin

In the absence of a competitive antagonist for Na channel-gate toxins which lacks intrinsic activity (cf. Sect. 3.10.2), the most specific way to test whether a positive inotropic agent acts by prolonging the open state of Na channels is to use the non-competitive antagonist TTX (or STX). TTX occludes Na channels and thereby prevents the effects of Na channel-gate toxins on membrane potential and Na and Ca influx. In guinea- pig papillary muscle, a TTX concentration which reduces ~max by 50% (10 #mol/litre) has no inhibitory influence on the positive inotropic effect of noradrenaline or theophylline and slightly inhibits that of dihydro-ouabain, but prevents the positive inotropic effect of half-maximally effective concentrations of cevadine, veratridine, and germitrine (Fig. 10). The fact that TTX fails to affect the positive inotropic effect of noradrenaline and theophylline is in agreement with the view that these agents act by increasing the cellular concentration of cAMP and that such an increase mediates the enhancement of slow inward Ca current and of the amount of Ca releasable from, and sequestered by an intracellular Ca store (for references see Scholz 1980). TTX lacks an effect on slow inward Ca current and is not expected to gain access to the intracellular space during extracellular application (Sect. 4.2.5). Furthermore, even if applied directly to the sarcoplasmic reticulum as in the experiments on skinned cardiac fibres by Fabiato and Fabiato (1973), TTX does not affect the Ca release-sequestration cycle as judged from the unchanged mechanical activity. The finding that TTX less effectively Cardioactive Substances that Prolong the Open State of Sodium Channels 59 inhibits the positive inotropic effect of dihydro-ouabain than that of ceveratrum alkaloids, although the action of both cardioactive steroids and Na channel-gate toxins depends on Na influx, has been discussed in Sect. 4.2.5. The use of TTX for the classification of inotropic mechanisms requires some precautions. It is necessary to rule out that the agent in question acts primarily on neural Na channels to cause the release of noradrenaline from intracardiac nerve endings. Such an effect is also blocked by TTX; in fact by concentrations much lower than those required to block sarcolemmal Na channels. Among the substances covered in this review only scorpion toxins were found to act mainly by a presynaptic mechanism. In the case of veratridine and cevadine, a neural effect is also demonstrable, but requires concentrations in excess of those affecting sarcolemmal Na channels (Sect. 4.2.5). It is also important to note that the pattern of inhibition by TTX depends on whether the Na channel-gate toxin acts reversibly or irreversibly. In the case of reversibly acting toxins, like veratridine (Honer]alger and Reiter 1975) or ATXII (Ravens 1976), TTX produces a stable inhibition if applied after the effects of the gate toxins have attained steady-state. With irreversibly acting toxins, like batrachotoxin (Honer]alger and Reiter 1977b), the effect of TTX, when applied in a submaximally effective concentration, is to delay the development, but not to prevent the effect of the Na channel-gate toxin. It appears that the change in cardiac membrane potential, such as the delay of the repolarization phase, requires only a small fraction of all Na channels to be modified (Horackova and Vassort 1973; Trautwein 1973). Thus TTX concentrations of 50 umol/litre or less, which reduce Vmax only up to 80% in guinea-pig papillary muscle (Baer et al. 1976), leave a significant fraction of Na channels unblocked, and this unblocked fraction of Na channels may suffice to mediate a prolongation of the action potential or decrease of the resting potential in the presence of a very high concentration of Na channel-gate toxin (Honer/iiger and Reiter 1977b). Ito et al. (1979) observed that 1 ~mol/litre TTX failed to restore the membrane potential in guinea-pig papillary muscle depolarized by the irreversibly acting palytoxin, and they conclude that "the partial antagonism by TTX implies the possibility that PTX [palytoxin] did not act 'specifically' on the Na channel and produced a leaky state". However, their findings that TTX delayed the onset of the palytoxin-induced depolarization and that reduction of [Na]o to 12.3 mmol/litre did restore the membrane potential in the presence of palytoxin are compatible with the view that palytoxin acts entirely by selectively opening Na channels. 60 P, Honerj/iger

6.3 Contraction Frequency

The frequency dependence of the positive inotropic effect of veratridine (Honerjiiger and Reiter 1975) or germitrine (Honer]ager and Reiter 1977a) differs from that of cAMP-increasing agents and, in certain details, from that of the cardioactive steroid dihydro-ouabain. The ceveratrum alkaloids lack a positive inotropic effect on the rested-state contraction (Sect. 4.2.5) whereas threshold effective concentrations at 1 Hz also increase the peak force of the rested-state contraction in the case of adrenaline and theophylline (cat papillary muscle; Bercsewicz and Reuter 1977) and noradrenaline and N 6 -2'-O-dibutyryl cAMP (guinea-pig papillary muscle; Seibel et al. 1978). Force-frequency relationships obtained with concentrations of dihydro-ouabain that are submaximally effective at 1 Hz (Ebner and Reiter 1977) are virtually identical to those obtained with veratridine or germitrine. Like the ceveratrum alkaloids, these concentrations ofdihydro-ouabain lack an effect on the rested-state contraction and produce a progressively increasing positive inotropic effect as the frequency is raised stepwise from 0.003 Hz to 0.5 Hz. Ceve- ratrum alkaloids lack a positive inotropic effect on the rested-state contraction even at concentrations exceeding the maximally effective concentration at a Hz by more than one order of magnitude (Sect. 4.2.5). In the cases of veratridine or cevadine, the demonstration of this lack of positive inotropic effect requires the elimination of their presynaptic effect (Sect. 4.2.5). In contrast, dihydro-ouabain increases the force of the rested-state contraction in concentrations that are maximally or supramaximally effective at 1 Hz (Ebner and Reiter 1977). In the cases of veratridine and germitrine, the phase of increased PNa is linked to the action potential, which explains the absence of a positive inotropic influence under resting conditions. However, it is conceivable that agents which increase resting PNa' like grayanotoxin I in rabbit atrial fibres (Seyama 1978), also increase the rested-state contraction.

6.4 Reduction of Extracellular Na Concentration

Reducing the extracellular Na concentration by one-half greatly diminishes the positive inotropic effect of cardioactive steroids, whereas the inotropic effect of catecholamines is unaffected (Reiter 1972a). Using this test, the ceveratrum alkaloids veratridine and cevadine were shown to be influenced in their effect on guinea-pig papillary muscle like the steroids (Reiter 1963), pointing to the importance of Na influx for the action of the alkaloids. Cardioactive Substances that Prolong the Open State of Sodium Channels 61

6.5 Variation ofExtracellular K Concentration

The level of the extracellular K concentration may influence the positive inotropic effect of Na channel-gate toxins by at least two different mechanisms, through its effect on transmembrane potential and on the activity of the Na-K pump. Ravens (1976) showed that ATXII failed to prolong the action potential and to produce a positive inotropic effect in guinea-pig papillary muscle if the K concentration was raised from 2.7 to 14.7 mmol/litre. The elevated K concentration caused a fall of the resting potential to about -50 mV, which is expected to inactivate normal Na channels (Beeler and Reuter 1970a). In contrast to ATXII, agents which increase the cellular cAMP concentration retain their positive inotropic effect and increase amplitude and duration of the action potential through their effect on Isi if Na channels are inactivated by depolarization (for references see Scholz 1980). Like the positive inotropic effect of ATXII, that of goniopora toxin on rabbit atrial preparations is prevented at an elevated (27 mmol/litre) extracellular K concentration (Fufiwara et al. 1979). Reducing the K concentration below 5.9 mmol/litre enhances the positive inotropic effect of veratridine (Honer]iiger and Reiter 1975). The associated hyperpolarization of the resting potential from about -80 mV at 5.9 mmol/litre [K]o has presumably little effect on the level of steady-state inactivation of the Na channels, since this seems to be already minimal at -80 mV (Chen et al. 1975). It seems more likely that the synergistic action of K withdrawal and veratridine is related to an inhibition of the Na pump by reduction of [K]o, which is expected to increase the extent of the veratridine-induced intracellular Na accumulation and therefore the positive inotropic effect. This test does not dis- crimiate between veratridine and dihydro-ouabain, the positive inotropic effect of which is likewise enhanced by a reduction in extracellular K concentration (Reiter et al. 1966). In contrast, the inotropic effect of adrenaline (Reiter et al. 1966) or theophylline (Scholz and de Yazikof 1971) is not increased by a lowering of extracellular K concentration.

6.6 Inhibition of the Na Pump by Dihydro-Ouabain

Dihydro-ouabain potentiates the positive inotropic effect of veratridine (Honer]iiger and Reiter 1975) and germitfine (Honer]iiger and Reiter 1977a), whereas veratridine and noradrenaline act additively. The increment in intracellular Na concentration induced by Na channel-gate toxins is normally counteracted by the Na-K pump, the activity of which is not directly influenced by Na channel-gate toxins. Hence, an inhibition of 62 P. Honerj~iger the Na pump will enhance the extent of the increment in intracellular Na concentration caused by agents which increase Na influx. The synergistic action of ceveratrum alkaloids and a specific Na pump inhibitor thus indirectly points to the critical role of intracellular Na concentration for the positive inotropic effect of Na channel-gate toxins.

6.7 Propranolol

(-+) Propranolol (5 /amol/litre) shifts the concentration-effect curve for the positive inotropic effect of veratridine to the right by a factor of 2 (Honerjdger and Reiter 1975). The same concentration of (+) propranolol causes the same degree of inhibition, although this isomer has only about 1/60 the activity of racemic propranolol in blocking the effects of /3-adrenoceptor activation (Howe and Shanks 1966). The antagonism is thus unrelated to the blockade of ~-adrenoceptors by propranolol, but is readily explained by the inhibitory effect of propranolol on myocardial Na channels which is identical for both isomers of propranolol (Pollen et al. 1969). Similarly, the antagonism of the positive inotropic effect of grayanotoxins by propranolol (10 gmol/litre, Sect. 4.5.2) may be entirely due to the blockade of Na channels by propranolol.

7 Summary and Conclusions

In recent years the detailed analysis of the mode of action of various Na channel-gate toxins has revealed that they prolong the open state of Na channels by mechanisms that differ in some detail. The alkaloids veratridine, batrachotoxin, and aconitine modify both activation and inactivation characteristics, while certain polypeptide toxins like ATXII predominantly affect Na inactivation and one of the scorpion venoms selectively modifies Na activation. These mechanisms were clarified by voltage-clamp experiments on single nerve fibres. Less direct experiments were performed on myocardial cells, but they all indicate that the Na channels of cardiac sarcolemma are also modified by various Na channel- gate toxins. The modification of cardiac Na channels results in a prolongation of the repolarization phase of the cardiac action potential which, depending on the particular toxin, may become evident at any potential level between the plateau and close to the resting potential. In embryonic cardiac cells, the application of Na channel-gate toxins revealed the presence of normally silent TTX-sensitive Na channels. Cardioactive Substances that Prolong the Open State of Sodium Channels 63

The prolonged phase of repolarization provides the depolarizing stimulus for coupled premature action potentials and thus explains the arrhythmogenic effect of Na channel-gate toxins. This type of arrhythmia is not specific for this kind of substances, but it is the consequence of a selective alteration of Na channel kinetics and therefore is completely inhibited by TTX. With the exception of aconitine, all Na channel-gate toxins with a demonstrable effect on myocardial Na channels were also shown to produce a positive inotropic effect in the prearrhythmic stage of action. Thus, a direct positive inotropic effect is observed in the presence of steroid alkaloids, diterpenoids, and polypeptides. Such a uniform response of the contractile mechanisms in the myocardial cell to substances of widely differing chemical structure suggests that the inotropic effect is somehow related to the known common mechanism of action of these toxins, i.e. the modification of the gating system of sarcolemmal Na channels. The experimental evidence surveyed indicates that the mechanism of the positive inotropic effect of Na channel-gate toxins involves the following chain of subcellular events: The initial step is the alteration of the sarcolemmal Na channels by the toxin. The essentiality of this step is deduced from the observation that TTX prevents or abolishes the positive inotropic effect at concentrations which also prevent or abolish the electrophysiologic changes resulting from the Na channel modification. The selectivity of action of TTX in blocking Na channels is well established. In addition, TTX was shown to cause comparatively little or no antagonism to the positive inotropic effect of drugs that do not affect Na channel kinetics (dihydro-ouabain, noradrenaline, theophylline). A cause-effect relationship requires that the causative event precedes the effect. This is clearly shown for Na channel-gate toxins in that the modified repolarization phase precedes the positive inotropic effect. The slowing of Na channel kinetics prolongs the time during which Na ions flow into the cell. In addition, the prolonged repolarization phase indirectly affects voltage- and time-dependent ionic currents other than Na current. The change in membrane potential, however, is not essential for the positive inotropic effect. As shown in voltage-clamp experiments on frog atrial trabeculae, the positive inotropic effect is still present if the prolongation of the action potential is prevented by submitting the preparation to depolarizations of constant amplitude and duration. Hence, the inotropic effect is coupled to the increase in Na current induced by the toxins. The importance of Na influx is also indicated by the fact that the positive inotropic effect of Na channel-gate toxins is potentiated by dihydro-ouabain, an inhibitor of Na efflux. 64 P. Honerj~ger

How does an increase in Na influx mediate a positive inotropic effect? A well-documented mechanism is the Na-Ca exchange, which provides an increase in transsarcolemmal Ca influx in response to an elevation of the intracellular Na concentration. In fact, Na channel-gate toxins were used to demonstrate the existence, in cardiac cells, of a Na-Ca exchange mechanism because they are convenient chemical tools to selectively produce an increase of the intracellular Na concentration in small cells.

Acknowledgments. The author gratefully acknowledges that his research on ceveratrum alkaloids was encouraged by Professor Dr. M. Reiter and Professor Dr. O. Krayer. I am indebted to Drs. P.F. Baker, O. Krayer, M. Lazdunski, M. Reiter, and W. Ulbricht for reading the manuscript and suggesting improvements.

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Factors Influencing Renal Sodium Reabsorption in Volume Expansion

FRANKLYN G. KNOX * and JOHN A. HAAS *

Contents

1 Introduction ...... 76

Renal Sodium Reabsorption ...... 76 2.1 Hemodynamic Factors ...... 76 2.1.1 Renal Blood Flow and Renal Vascular Resistance ...... 76 2.1.2 Intrarenal Distribution of Blood Flow ...... 77 2.2 Glomerular Filtration Rate ...... 79 2.2.1 Effect of Changes in GFR ...... 79 2.2.2 Intrarenal Distribution of GFR ...... 80 2.3 Physical Factors ...... 82 2.3.1 Plasma Oncotic Pressure ...... 82 2.3.2 Peritubule Capillary Hydrostatic Pressure ...... 85 2.3.3 Interstitial Pressure ...... 86 2.3.4 Plasma Sodium Concentration ...... 88 2.3.5 Preexisting Extracellular Fluid Volume ...... 89 2.3.6 Tubule Geometry ...... 90 2.4 Segmental Sodium Reabsorption ...... 90 2.4.1 Acute ECVE ...... 90 2.4.2 Chronic ECVE ...... 93 2.5 Hormonal Factors ...... 94 2.5.1 Renin-Angiot ensin-Aldostcrone System ...... 94 2.5.2 Natriuretic Hormone ...... 95 2.5.3 Prostaglandins ...... 97 2.5.4 Antidiuretic Hormone ...... 97 2.5.5 Parathyroid and Thyroid Hormone ...... 97 2.5.6 Neurohumoral ...... 98 2.6 Response of the Perinatal Kidney to Volume Expansion ...... 99 2.7 Gastrointestinal and/or Hepatoportal Control of Renal Sodium Excretion ...... 101

3 Summary ...... 102

References ...... 103

* Nephrology Research Laboratories, Departments of Physiology and Biophysics and Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, 55905, USA 76 F.G. Knox and J.A. Haas

1 Introduction

This review discusses factors which influence renal sodium reabsorption in volume expansion. The factors are considered under seven headings: hemodynamic factors; glomerular filtration rate; physical factors; segmental sodium reabsorption; hormonal factors; response of the perinatal kidney to volume expansion; and gastrointestinal or hepatoportal control of renal sodium excretion. The results discussed consist primarily of clearance, micropuncture, microperfusion, and histologic studies. It seems justified to inject a word of caution in regard to interpretation of the data contained herein. In the majority of the studies, the animals were anesthetized, subjected to varying degrees of surgical trauma, and subsequently given an extracellular volume expansion (ECVE). Inasmuch as these maneuvers are not physiologic, extrapolation of the results to normal renal function should be done with caution.

2 Renal Sodium Reabsorption

2.1 Hemodynamic Factors

2.1.1 Renal Blood Flow and Renal Vascular Resistance

Expansion of the extracellular fluid volume (ECFV) by an infusion of isotonic saline has been shown to decrease renal vascular resistance and increase renal blood flow (Barger et al. 1961 ; Earley and Friedler 1965a; Wallin et al. 1971). In initial studies, the extent to which this increased renal blood flow alone results in increased sodium excretion was evaluated in clearance experiments in dogs by infusing vasodilators alone, or in the presence of a saline load (Earley and Friedler 1965b). The results of these studies demonstrated that in hydropenic animals, increasing renal blood flow by vasodilators alone was accompanied by an increased sodium excretion in spite of an unchanged filtered load. In animals receiving a saline load, increased renal blood flow of the same magnitude was associated with a threefold greater natriuresis. Further studies demonstrated that infusion of iso-oncotic Ringer's solution, designed not to alter plasma protein concentration, resulted in large increases in arterial pressure, renal blood flow, and urinary sodium excretion. If the kidneys were vasodilated with acetylcholine prior to the iso-oncotic Ringer's loading, the additional natriuresis seen with the vasodilated kidney was significantly reduced (Martino and Earley 1967). These studies were taken as evidence that increases in renal blood flow, per se, can increase Factors Influencing Renal Sodium Reabsorption in Volume Expansion 77 sodium excretion, but can only partially account for the natriuresis accompanying saline loading. The possibility exists, however, that the natriuretic effect seen with vasodilators is due to a direct action of the drug on the renal tubule and that the increased renal blood flow per se is not necessarily natriuretic. For example, secretin is a vasodilator which is not natriuretic (Marchand et al. 1977a). Further, small infusions of saline which increase urinary sodium excretion may not result in increases in renal blood flow (Martino and Earley 1967).

2.1.2 Intrarenal Distribution of Blood Flow

Volume expansion may be associated with increased medullary renal blood flow, since increased sodium excretion is accompanied by a decrease in urinary osmolality and the extraction ratio ofp-aminohippurate (PAH) (Earley and Friedler 1964, 1965a; Friedler and Earley 1966). This decrease in urinary osmolality, which could not be accounted for by changes in either antidiuretic hormone (ADH) or solute excretion, and the assump- tion that PAH is extracted only from blood perfusing the cortex, led Earley and Friedler to hypothesize that saline loading in the dog results in an increased medullary blood flow, and that there may be a causal relationship between this intrarenal distribution of blood flow and the associated natriuresis of ECVE. The issue of whether a change in the intrarenal distribution of blood flow is associated with the renal vasodilation seen with volume expansion has subsequently been studied with a variety of techniques. The different techniques used to measure intrarenal blood flow distribution include the use of extractable substances such as PAH, dye dilution, washout of inert gases like s s Kr and 133 Xe, H~ clearance, glomerular basement-membrane antibody technique, and radioactive-labeled microspheres. A description of the different methods and a discussion of their validity is given by Barger and Herd (1973). Initial studies which utilized the inert-gas washout or H2 clearance method to evaluate intrarenal distribution of blood flow reported that in volume expanded dogs, blood flow in the renal cortex increases, but there was not a significant change in the distribution of blood flow within the cortex (Munck et al. 1970; Loyning 1974). This finding was essentially confirmed in a subsequent study also carried out in dogs. Bovee and Webster, utilizing the inert-gas washout method and PAH extraction, reported that the increase in renal blood flow during moderate saline loading is limited to the cortex, but during large saline loads, cortical and noncortical flows increase proportionately (Bovee and Webster 1971). These findings are in agreement with recent observations of Velasquez, who applied the dye-dilution technique for quantitation of intrarenal blood flow distribution, and reported that during the early phase of saline 78 F.G. Knox and J.A. Haas diuresis renal vasodilatation is confined to the cortex, but during later stages of saline loading noncortical blood flow increases also ( Velasquez et al. 1973, 1975). Interestingly, a study in the rat utilizing the X e washout method found no significant change in either total renal blood flow or outer cortical blood flow after volume expansion with saline (Kinney and DiScala 1974). More recent results evaluating intrarenal distribution of blood flow during ECVE have been obtained with radioactive microspheres. These studies have shown that, in the dog, an intrarenal distribution of blood flow to inner cortical nephrons occurs (Blantz et al. 1971;Stein et al. 1972; Bruns et al. 1974). Surprisingly, in the rat, an intrarenal distribution of blood flow to the superficial cortex following volume expansion has been reported (Wallin et al. 1971). However, this discrepancy was attributed to an axial streaming of the microspheres caused by the renal vasodilation and reduced hematocrit accompanying volume expansion. The mechanism of cortical blood flow distribution during acute saline loading in the dog has been examined with microspheres (Migdal et al. 1975); their data showed that hemodilution without volume expansion or with intravascular volume expansion alone resulted in a distribution of blood flow to the superficial cortex. This superficial distribution of blood flow was caused by a decreased hematocrit. During saline loading, distribution of blood flow to the inner cortex occurred, which was hypothesized to be a consequence of expansion of the intrarenal interstitial volume. Two mechanisms have been proposed whereby increased blood flow to inner cortical nephrons could affect renal handling of sodium during volume expansion. The first is the medullary washout hypothesis of Earley and Friedler (1965a,b). They proposed that saline loading produces an increase in inner cortical and presumably medullary blood flow. This causes a reduction in medullary interstitial hypertonicity, thus reducing the osmotic movement of water out of the descending limb. A larger volume of tubular fluid with the same amount of sodium, but at a lower concentration, would enter the water impermeable ascending limb. Reabsorption of sodium to the same minimal concentration in the ascending limb would result in the delivery of a larger volume of fluid and an increased total amount of sodium beyond the loop of Henle. This alteration would be most marked in the inner cortical nephrons whose loops descend into the medulla. Another mechanism whereby intrarenal distribution of blood flow may modulate the natriuresis of ECVE is that it could result in a selective decrease in the filtration fraction in inner cortical nephrons and con- sequent reduction in proximal sodium reabsorption. This hypothesis assumes a direct relationship between peritubular capillary protein oncotic pressure and proximal sodium reabsorption. This hypothesis is based on Factors Influencing Renal Sodium Reabsorption in Volume Expansion 79 experimental observations in several different species. Perhaps the most convincing was a series of experiments in which Nissen made use of the unique blood supply to the kidney of the cat (Nissen 1968). He calculated the filtration fraction from the postglomerular circulation by comparing the plasma concentrations of inulin and protein in the blood leaving the two venous drainage areas with the concentrations in cortical blood. He observed a greater fall of the filtration fraction in the inner cortex than in the outer cortex after volume expansion. Micropuncture studies in the rat by Daugharty et al. (1972), who calculated superficial nephron filtration fraction (SNFF) from systemic and efferent arteriolar protein concentrations, and Barratt et al. (1973), who calculated SNFF by means of the antiglomerular basement membrane antibody technique, showed that the filtration fraction was reduced to a greater extent in the whole kidney compared to the superficial cortex during Ringer loading. Likewise in dogs, using the Hanssen technique and microspheres, estimated superficial nephron filtration fraction remained unchanged after volume expansion, but juxtamedullary filtration fraction decreases significantly (Bruns et al. 1974).

2.2 Glomerular Filtration Rate

2.2.1 Effect of Changes in GFR Associated with increases in renal blood flow, several studies have shown that glomerular filtration rate (GFR) is elevated by ECFV expansion (de Wardener et al. 1961; Levinsky and Lalone 1963; Davidrnan et al. 1972). This observation is important as urinary sodium excretion represents the difference between the filtered load of sodium and subsequent reabsorption by the tubules. Therefore, a parallel change in filtered load and sodium excretion seen following volume expansion could, theoreti- cally, explain the rise in sodium excretion seen following volume expansion, without necessarily invoking changes in tubular reabsorption. How- ever, de Wardener et al. showed that saline infusion increased urinary sodium excretion independent of changes in GFR. In these studies, GFR was prevented from increasing by simultaneously decreasing the perfusion pressure to the kidney (de Wardener et al. 1961). This observation was subsequently confirmed by a number of other investigators who also demonstrated that when ECFV is acutely increased in the presence of fixed adrenocortical activity, sodium excretion increased independently of changes in GFR (Levinsky and Lalone 1963;Rector et al. 1964). 80 F.G. Knox and J.A. Haas

2. 2. 2 Intrarenal Distribution of GFR

Whereas increases in GFR may not entirely account for the natriuresis of ECVE, the relationship between changes in ECFV and distribution of single neprhon glomerular filtration rate (SNGFR) may be important as it has been proposed that a redistribution of filtrate may modify urinary sodium excretion. Intrarenal distribution of filtrate before and after volume expansion has been evaluated with a number of techniques. The first is an indirect evaluation and involved comparing superficial SNGFR to total kidney glomerular filtration rate. The results are conflicting, with some studies reporting equal changes in whole kidney GFR and superficial SNGFR after volume expansion (Bartoli and Earley 1971; Mandin et al. 1971; Daugharty et al. 1972; Davidman et al. 1972), while others found redistribution of filtrate to outer cortical nephrons (Gertz et al. 1967; Barratt et al. 1973). These data should be critically evaluated for several reasons. First, an increased distribution of filtrate to outer cortical nephrons was found when a previously punctured tubule was repunctured during acute volume expansion (Mandin et al. 1971; Stein et al. 1972). In contrast, no change in the intrarenal distribution of filtrate was found when a freshly punctured tubule was utilized. This difference has been atrributed to artifactual alterations in intratubular dynamics in the repunctured tubule due to the high tubular pressure associated with volume expansion, the so-called recollection artifact. However, in sharp contrast, a similar artifact was not noted in other studies in either the dog or rat, in which ECVE caused a proportional increase in both SNGFR and GFR (Brenner et al. 197 la; Schneider et al. 1972). Second, these results involve comparison of only superficial SNGFR and total kidney GFR, without direct measurement of SNGFR in deep nephrons. It has also been proposed that the disagree- ment between the results concerning the changes in SNGFR distribution following volume expansion could be explained on the basis of the degree of volume expansion. Herrera-Acosta concluded that a 10% body weight volume expansion did not change intrarenal filtrate distribution, whereas a progressive volume expansion to 15% of body weight results in a dis- proportionate increase in the filtration rate of superficial nephrons in comparison to total kidney GFR (Herrera-Acosta et al. 1972). Another method used to study the distribution of GFR is the ferrocyanide technique. This method involves administering ferrocyanide, which distributes into the extracellular fluid volume and behaves like a glomerular indicator. After injection, the pedicle is ligated, the kidney removed and microdissected. The distance between the glomerulus and the distal front of the precipitated bolus is assumed to be proportional Factors Influencing Renal Sodium Reabsorption in Volume Expansion 81 to the nephron filtration rate. The advantage of this method is that it allows for a more quantitative estimate of the ratio of both superficial and deep SNGFR. Although some studies do not find a change in intrarenal distribution (Bruns et al. 1974; Coelho 1974), others report an increase in the superficial distribution of SNGFR after either chronic or acute salt loading when the labeled ferrocyanide is given as a pulse injection, i.e., Hanssen's technique (de Rouffignac and Bonvalet 1970; Baines 1973). However, when the glomerular marker is infused, SNGFR increases proportionately in both superficial and juxtamedullary nephrons (Carriere et al. 1972; Coelho 1973; Clausen and Tyssebotn 1973; Poujeol et al. 1975). This discrepancy was studied by Charbardes, and it was concluded that the apparent change in intrarenal distribution during ECVE observed with pulse injection is probably the result of an artifactual streaming caused by the method of administration of the label (Chabardes et al. 1974). In addition, a redistribution of SNGFR after acute volume expansion using Hanssen's technique was found in young rats, but not in mature adults (Baines 1973). These studies suggest that structural changes with age may modify the response of SNGFR distribution to volume expansion. Finally, two early micropuncture studies examined directly, by micropuncture of both superficial and juxtamedullary nephrons, the effect of ECVE on the distribution of single nephron filtration rate in these nephron populations (Horster and Thurau 1968; Jamison and Lacy 1971). Both studies showed a shift of SNGFR distribution toward the superficial nephrons. However, the physiologic significance of a change in the intrarenal distribution of SNGFR is speculative without knowing whether actual sodium transport is altered in these nephron populations during ECVE. In the acute volume expansion study in which sodium concentration in tubule fluid was measured along with inulin, sodium reabsorption was significantly reduced in superficial nephrons, and did not change in deep nephrons (Jamison and Lacy 1971). In contrast, a more recent study revealed that delivery of sodium to the bend of the loop of Henle in the deep nephrons was greater than estimates of delivery in superficial nephrons during Ringer loading (Osgood et al. 1978). The hypothesis that the intrarenal distribution of single nephron glomerular filtration rate can affect urinary sodium excretion came from studies by Barger, who proposed that because the juxtamedullary or deep nephrons have long loops of Henle, it is possible that more sodium is removed from them than superficial nephrons with their short loop of Henle (Barger 1966). Thus, intrarenal distribution of glomerular filtration may modify sodium excretion during ECVE by shifting from deep "salt-saving" nephrons to superficial "salt-losing" nephrons. However, a micropuncture study in the Rhesus monkey, a species with short loops of Henle, shows that the fraction of filtered sodium arriving in the early 82 F.G. Knox and J.A. Haas distal tubule is similar to values reported for the dog, a species with long loops of Henle (Bennett et al. 1968). This would argue against the concept that the length of the loop of Henle is an important factor in the magnitude of sodium transport.

2.3 Physical Factors

2. 3.1 Plasma Oncotic Pressure

On the basis of clearance studies in the dog, it was concluded that net fluid reabsorption is determined, in part, by the rate of removal of reabsorbate by the capillary circulation (Martino and Early 1967). The rate of removal of this reabsorbate is, in turn, dependent upon the net balance of colloid osmotic and hydrostatic pressures across the peritubular capillary wall. Evidence was presented supporting the hypothesis that these "physical forces" play an important role in the decreased proximal tubular reabsorption and increased sodium excretion seen in response to volume expansion. It was demonstrated that a saline infusion which depressed plasma protein concentration, without increasing arterial pressure or renal blood flow, increased urinary sodium excretion. Subsequent restoration of plasma protein concentration by systemic infusion of hyperoncotic albumin decreased sodium excretion in spite of an increase in arterial pressure and renal blood flow (Martino and Earley 1967). This observation was taken as evidence that depressed plasma protein is a major determinant of the natriuresis seen with ECVE. Subsequent micropuncture and microperfusion studies, in a variety of experimental conditions designed to assess the importance of plasma oncotic pressure by controlling other physical forces, presented data consistent with the postulate that changes in postglomerular capillary oncotic pressure play a significant role in the regulation of proximal sodium reabsorption (Lewy and Windhager 1968; Wind- hager et al. 1969; Spitzer and Windhager 1970). Brenner, utilizing both free-flow micropuncture and microperfusion techniques, demonstrated a direct relationship between oncotic pressure in efferent arterioles and absolute reabsorption by the proximal tubule of the rat (Brenner et al. 1969b; Brenner and Troy 1971). The relationship between peritubular capillary oncotic pressure and proximal sodium reabsorption has also been studied in the volume expanded animal. During volume expansion, Brenner et al. (1971b) found that capillary microperfusion with hyperoncotic albumin solution increased reabsorption by the proximal tubule and, similarly, Knox found that infusion of hyperoncotic albumin solution in the renal artery increased proximal sodium reabsorption in saline Factors Influencing Renal Sodium Reabsorption in Volume Expansion 83 loaded dogs (Knox et al. 1973). In micropuncture studies in rats, Daugh- arty compared the effects of iso-oncotic and colloid-free volume expansion on proximal reabsorption (Daugharty et al. 1972). In rats in which the filtered load was kept constant by means of aortic constriction, volume expansion with colloid-free (Ringer's) solution resulted in a significant decrease in proximal reabsorption which was associated with a marked reduction in peritubule protein concentration. In contrast, iso-oncotic infusions resulted in no significant fall in proximal reabsorption and little change from control in peritubule protein concentration. Son- nenberg and Solomon, using free-flow micropuncture, observed that relative to control hydropenic values, extracellular volume expansion with colloid-free Ringer's solution resulted in a significant decrease in proximal reabsorption, whereas following comparable infusions of iso-oncotic fluid, no significant reduction could be detected (Sonnenberg and Solo- mon 1969). Several investigators, however, have failed to find a relationship between peritubule capillary oncotic pressure and reabsorption by the proximal tubule. Specifically, in the absence of saline loading, two studies found no difference in proximal sodium reabsorption between iso-oncotic perfusate versus oncotic-free perfusate (Rumrich and Ullrich 1968; Conger et al. 1973). Further, microperfusion studies showed that reductions in absolute reabsorption induced by saline infusion were quantitatively similar in tubules exposed to the reduced peritubule capillary oncotic pressure and in microperfused tubules with constant peritubular oncotic pressure (Holzgreve and Schrier 1975). Additionally, Kuschinsky et al. compared proximal fractional reabsorption and plasma protein concentration in rats acutely or chronically infused with isotonic saline (Kuschinsky et al. 1970). During acute ECVE, proximal fractional reabsorption (FR) was reduced from 61% to 42%, concomitant with a reduced plasma protein from 5.8% to 5.2%. However, during chronic saline infusion, fractional reabsorption (FR) was only slightly decreased to 52%, not statistically different from a control group of animals in which FR was 57%. In these two groups plasma protein concentration was reduced to a greater extent (from 5.9% to 3.6% in the control and chronically infused annimals, respectively). Ott et al. investigated the effect of increased peritubule capillary oncotic pressure on sodium reabsorption by the proximal tubule in hydropenic and volume expanded dog (Ott et al. 1975). While efferent oncotic pressure during albumin infusion was increased to the same degree in both groups, proximal sodium reabsorption was increased only in the volume expanded animals. The results suggested that ECVE altered the effect of increased peritubule oncotoc pressure on sodium reabsorption by the proximal tubule. It was speculated that increased permeability of the proximal tubule following saline loading 84 F.G. Knox and J.A. Haas allowed for changes in peritubule capillary oncotic pressure to affect proximal reabsorption. Several studies support a role for altered permeability of the proximal tubule following ECVE. In electrophysiological studies, changes in permeability of the proximal tubule following saline loading in the necturus have been demonstrated (Bentzel et al. 1970; Boulpaep 1972; Maunsbach and Boulpaep 1980). Based on these data, it seems likely that increases in membrane permeability are a prerequisite for increased peritubule oncotic pressure to result in increases in isotonic reabsorption by the renal proximal tubule. Arterial hematrocrit is another physical factor which significantly influences urinary sodium excretion during ECVE. Knox et al. found in micropuncture experiments in dogs that acute reductions in arterial hematocrit were associated with a decrease in fractional reabsorption by the proximal tubule, despite maintenance of a normal extracellular fluid volume and no significant change in urinary sodium excretion (Knox et al. 1968). Clearance experiments have demonstrated a direct relationship between sodium excretion and blood viscosity during volume expansion (Schrier et al. 1970). Further studies in which hematocrit was either increased or decreased in both hydropenic or volume expanded animals, demonstrated that acute changes in hematrocrit significantly affect sodium excretion and renal hemodynamics in both conditions (Schrier and Earley 1970). Increasing hematocrit during hydropenia or ECVE decreased electrolyte excretion and renal blood flow. Decreasing hematocrit during hydropenia was associated with an increase in electrolyte excretion. The effect of increasing or decreasing arterial hematocrit on proximal sodium reabsorption has been investigated in dogs (Burke et al. 1971). It was demonstrated that fractional reabsorption by the proximal tubule can be increased or decreased by acutely increasing or decreasing the hematocrit. It seems likely that the effects of arterial hematocrit are mediated through changes in blood viscosity and subsequent effects on peritubule capillary hydrostatic pressures. It has also been demonstrated that after anesthesia and surgery animals have a markedly impaired ability to excrete a saline load. Keck and co- workers reported that this phenomenon was due to an increased hematocrit, probably due to an increased permeability of the capillaries which results in a large extravasation of protein and fluid into the extravascular space (Keck et al. 1973). This observation was confirmed in the rat by Maddox, who reported a significant increase in hematocrit accompanied by a large fall in plasma volume following surgery (Maddox et al. 1977). Extracellular volume expansion has also been shown to depress net absorption of sodium and water by rat (Richet and Hornych 1969; Humphreys and Earley 1971), dog (Higgins and Blair 1971), cat (Gutrnan and Benzakein 1970), and rabbit (DiBona et al. 1974)small intestine. Factors Influencing Renal Sodium Reabsorption in Volume Expansion 85

From studies in the cat, it was postulated that ECVE reduces primarily the lumen to blood sodium flux, analogous to the process of tubular reabsorption in the kidney (Gutman and Benzakein 1970). On the other hand, two studies in the dog (Higgins and Blair 1971; I~Iumphreys and Earley 1971), one in the rat (Chanard et al. 1976), and one in the rabbit (DiBona et al. 1.974) found that ECVE leads to an increase in blood to lumen sodium flux. The mechanism whereby ECVE depresses intestinal sodium and water transport has been investigated. The results indicate an important role for physical factors in modulating intestinal transport. In vitro studies in dog jejunum showed that when serosal hydrostatic pressure was increased by 2-6 cm H~ O, fluid absorption was stopped, and further increases in serosal pressure resulted in fluid secretion (Hakim and Lifson 1969). In vitro studies in rats demonstrated that capillary hydrostatic pressure and colloid osmotic pressure exert opposite effects on transjejunal water transport (Lee 1973). Further, it has been reported that during ECVE in vivo, the decreased net absorption of sodium is accompanied by an increased permeability of the intestinal mucosa (Humphreys and Earley 1971 ). It has also been demonstrated that saline infusion results in a dis- tension of intercellular spaces in the rabbit jejunum (DiBona et al. 1974). In addition, in vivo studies suggest that intestinal absorption increases when plasma oncotic pressure is increased by albumin infusion (Humph- reys and Earley 1971). These observations imply that capillary absorption plays a role in intestinal absorption and that permeability changes modulate the net absorption of sodium and water, as may be the case for fluid reabsorption by the renal proximal tubule during ECVE.

2.3.2 Peritubule Capillary Hydrostatic Pressure

Earley et al., on the basis of clearance studies in dogs, concluded that the natriuretic effect of renal vasodilatation, induced either by infusion of vasodilators, plasma or iso-oncotic saline infusion, may in part result from decreased tubular reabsorption as a consequence of the transmission of the increased pressure to the peritubular capillaries (Earley and Friedler 1966; Earley et al. 1966). It also has been proposed that the exaggerated natriuresis of hypertensive patients and rats when given a saline infusion may be due to a direct effect of the increased arterial pressure to decrease fractional reabsorption of sodium (Papper et al. 1960; Buckalew et al. 1969; Stumpe et al. 1970). In addition, a direct relationship between the natriuresis of volume expansion and renal perfusion pressure has been reported from studies in the isolated dog kidney (McDonaM and de Wardener 1965). Bank and co-workers, using split-drop techniques in saline loaded rats, reported that when renal perfusion pressure was 86 F.G. Knox and J.A. Haas lowered to 70-90 mmHg, the reduction in fractional reabsorption which usually occurs in the proximal tubule was greatly minimized (Bank et al. 1969). Associated with this finding was a concomitant decrease in urinary sodium excretion. From these results the authors suggested that the changes in proximal reabsorption during saline loading are related to the increase in peritubular hydrostatic pressure. In an attempt to determine whether transmitted increments in capillary hydrostatic pressure are related to decreases in sodium reabsorption during volume expansion, deep intrarenal venous pressure was used as a qualitative index of renal capillary pressure (Martino and Earley 1968). It was concluded, based on correlations between changes in intrarenal venous pressure and sodium excretion following either saline or plasma infusion, that increases in peritubular capillary hydrostatic pressure inhibit reabsorption. Stop-flow microperfusion with or without combined local microperfusion of peritubular capillaries has shown that, while hydrostatic pressure from the lumen is not an effective driving force for fluid reabsorption, increases in hydrostatic pressure from the contraluminal side markedly inhibit fluid reabsorption (Sato 1975). Finally, Kunau et al. showed that in rats under- going a saline diuresis an acute increase in renal perfusion pressure resulted in a marked increase in sodium excretion (Kunau and Lameire 1976). This natriuresis occurred in the absence of a change in either total GFR or superficial SNGFR. It was also observed that the natriuresis was not the result of an increased sodium delivery from the superficial late distal tubule. This led to the conclusion that it must be related to an inhibition of sodium reabsorption in either the collecting system or in deep nephrons.

2. 3. 3 Interstitial Pressure

Clearance experiments in dogs demonstrated that administration of dextran in isotonic saline leads to a natriuresis, whereas dextran in glucose does not (Schrier et al. 1968). Dextran in saline was associated not only with increased plasma volume, but also with increased interstitial volume, whereas dextran in glucose increased plasma volume alone. From this observation it was postulated that without expansion of the interstitial space, intravascular volume expansion may not be a sufficient stimulus to increase sodium excretion. Micropuncture techniques have been used to study the respective influence of each of the Starling forces upon proximal tubular reabsorption during hydropenia and volume expansion. The Starling forces, hydrostatic and oncotic pressures across the peritubular capillary wall, presumably mediate capillary uptake and passive backflux into the proximal tubular lumen through changes in renal interstitial pressure. Peritubular capillary uptake is determined not only by the balance of the Starling Factors Influencing Renal Sodium Reabsorption in Volume Expansion 87 forces, i.e., the net reabsorption pressure, but also by the permeability surface area of the peritubular capillary. Thus, changes in either para- meter lead to changes in capillary uptake. The relationship between net reabsorption pressure and the permeability surface area of the peritubular capillary (reabsorption coefficient) has been studied in the volume expanded rat with differing results. Tucker and Blantz measured proximal reabsorption in rats during hydropenia and saline expansion. They concluded that changes in proximal reabsorption correlate best with changes in filtered load (Tucker and Blantz 1978). Quinn and Marsh concluded that it was unlikely the reabsorption coefficient was different between hydropenia, Ringer's expansion, and plasma volume expansion (Quinn and Marsh 1979). They found a strong correlation between absolute proximal reabsorption and net interstitial pressure, which was taken as evidence for a role for interstitial pressure in regulating proximal reabsorption. However, interstitial pressure was directly measured only in saline loaded animals and was calculated for the other conditions. Stron- ger evidence for a positive correlation between renal interstitial pressure and sodium excretion during volume expansion has been presented by Marchand, who observed that reductions in renal perfusion pressure by renal artery constriction prior to volume expansion blocked both the increase in renal interstitial pressure as well as the increase in sodium excretion (Marchand 1978). Kallskog and Wolgast calculated peritubular protein concentration and measured interstitial space pressure (Kallskog and Wolgast 1973). In saline expansion, the interstitial hydrostatic pressure almost doubled, whereas interstitial oncotic pressure fell to almost zero. They concluded that the peritubular capillary has a large and a small pore system, and further postulated that the effective capillary area increases during saline loading due to expansion of the capillary pores. Experiments providing evidence that intrarenal factors are the initial determinants of the natriuresis of saline loading were performed by Fitz- gibbons et al. (1974). As mentioned previously, the natriuretic effect of saline loading has been demonstrated to persist in spite of a reduction of GFR to below preexpansion levels (de Wardener et al. 1961). However, Fitzgibbons reported that in the rat, reduction of renal artery pressure prior to the initiation of ECVE virtually abolished the natriuretic effect of volume expansion. From these findings, they hypothesized that ECVE can induce a natriuresis only if the kidney has been exposed to at least a transient increase in either interstitial hydrostatic pressure and/or renal plasma flow. Further, the results gave strong evidence against an important role for a reduction in plasma oncotic pressure or a circulating hormone as mediators of the natriuresis. This finding was confirmed in a micropuncture study in the rat by Osgood which demonstrated that volume expansion did not decrease proximal reabsorption or cause an 88 F.G. Knox and J.A. Haas increase in urinary sodium excretion in immediate-clamp rats (Osgood et al. 1977). In contrast, comparable aortic clamping 40 rain after initiation of volume expansion did not prevent a fall in proximal reabsorption or an increase in urinary sodium excretion.Marchand evaluated the effect of reduced peritubule capillary oncotic pressure at reduced renal artery pressure on renal interstitial pressure and sodium excretion in the dog (Marchand 1978). He found that reducing renal artery pressure before volume expansion reduces or prevents increases in interstitial pressure and fractional sodium excretion, depending on the magnitude of reduction of renal artery pressure. Studies, also performed in dogs, demonstrated a saline-induced natriuresis, although blunted, even though perfusion pressure was reduced prior to the accompanying volume expansion (Klemmer et al. 1978). This study suggested a possible species difference in the renal natriuretic response to volume expansion at reduced renal perfusion pressure between dogs and rats. In order to test the hypothesis that these conflicting results could be due to differences in preexpansion basal sodium excretion, Lameire manipulated the dietary salt intake to adjust preexpansion fractional excretion of sodium (FENa %) to the same level in both species (Lameire et al. 1979). Hydropenic or salt depleted dogs showed a small but significant increase in FENa % following a 7.5% body wt. volume expansion. In the rat, however, a significant natriuresis did not occur unless the rats were salt loaded. These results were interpreted to mean that an appropriate intrarenal environment is necessary in the rat, but not the dog, for volume expansion to result in a significant natriuresis. Further, it was concluded that the natriuretic response to ECVE was not dependent on the moment of lowering the renal perfusion pressure. However, it should be emphasized that in both species, regardless of preexpansion basal sodium excretion, prior reduction of renal perfusion pressure markedly attenuated the magnitude of the natriuresis in response to volume expansion.

2. 3. 4 Plasma Sodium Concentration

It has been shown that following infusion of hypertonic saline there are increases in plasma sodium concentration, filtered sodium, and urinary sodium excretion (Kamm and Levinsky 1964). The difficulty in assessing the importance of plasma sodium concentration, per se, as a mediator of the natriuresis following hypertonic saline infusion is that, besides hypernatremia, this experimental maneuver also leads to expansion of the ECFV and a fall in plasma oncotic pressure, all of which could by themselves result in increased sodium excretion. In order to circumvent this problem, Kamm and Levinsky infused hypertonic saline directly into one renal artery of a dog. Despite a decreased filtered load below that of Factors Influencing Renal Sodium Reabsorption in Volume Expansion 89 preinfusion levels and a comparable volume expansion in the contralateral kidney, the results showed an ipsilateral increase in sodium excretion, which was attributed to an effect of hypernatremia per se to depress net tubular reabsorption of sodium (Kamm and Levinsky 1965). In order to determine whether a low plasma sodium could depress the natriuretic response to ECVE, Davis compared fractional sodium reabsorption in the proximal tubule in normal dogs and in dogs made hyponatremic by chronic administration of vasopressin, water, and ethacrynic acid. Hypo- natremic dogs expanded with hypotonic saline had a smaller decrease in fractional reabsorption than normal dogs expanded with an equal amount of isotonic saline (Davis et al. 1970). Experiments in dogs, which were infused with equal amounts of sodium chloride per kilogram of body weight as either hypotonic, isotonic, or hypertonic solutions, demonstrated that in spite of the fact that hypotonic saline led to a greater expansion of the blood volume and interstitial space, sodium excretion was significantly less in this group (Schrier et al. 1969). Thus, these studies suggest that plasma sodium concentration may influence the natriuretic response to volume expansion.

2.3.5 Preexisting Extracellular Fluid Volume

The effect of the level of the preexisting extracellular fluid volume on the response to acute volume expansion has been studied in the dog and rat. In the dog, animals on a high salt intake and deoxycorticosterone acetate (DOCA) administration have a greater diuretic and natriuretic response to either hyperoncotic albumin solution (Knox et al. 1970) or isotonic saline loading (Higgins 1971). However, in ratsaparadoxical effect of dietary salt intake on the renal response to hypertonic saline loads has been reported (Ben-Ishay 1973). Animals on a low salt regimen demonstrated an enhanced diuretic and natriuretic response to hypertonic saline loading compared with rats on a high salt diet. In contrast, studies in dogs maintained on a high sodium intake revealed that they did not have a significantly different natriuresis from those on a low sodium intake, following hypertonic saline infusion (Diaz-Buxo et al. 1976). This was also found to be the case whether or not dogs were in positive or negative sodium balance. However, the natriuretic response to isotonic saline loading was smaller in dogs in negative sodium balance compared with those in positive sodium balance. These findings suggest that hypernatremia accompanying hypertonic saline infusion can override factors promoting sodium retention in sodium depleted dogs, but does not further augment sodium excretion in states of positive sodium balance. 90 F.G. Knox and J.A. Haas

2. 3. 6 Tubule Geometry A mechanism for glomerular-tubular balance was suggested by Gertz and others (Gertz et al. 1965; Brunner et al. 1966). In this proposal, the rate of proximal tubular reabsorption is thought to be proportional to intratubular volume, as a result of a direct relationship between tubular surface area and reabsorptive rate. Changes in filtration rate result in proportional changes in tubular surface area and subsequently sodium reabsorption. It was proposed that the mechanism by which ECVE suppresses proximal reabsorption was the result of two factors, a decrease in intrinsic reabsorptive capacity and a diminished tubular volume per given level of GFR (Rector et al. 1967). However, there is experimental evidence which indicates that tubule geometry does not significantly influence sodium reabsorption. Microperfusion studies, which changed either tubular volume or radius, showed no significant correlation between reabsorption and tubular shape or size (Morgan and Berliner 1969b; Burg and Orloff 1968).

2.4 Segmental Sodium Reabsorption

2.4.1 Acute ECVE There is much evidence from micropuncture studies in the dog and rat to indicate that the natriuresis of acute ECVE is associated with decreased sodium reabsorption by the renal tubule. Numerous studies have demonstrated that ECVE significantly decreases sodium reabsorption by the proximal tubule (Rector et al. 1964; Dirks et al. 1965; Cortney et al. 1965). Because the resulting increase in sodium delivery from the proximal tubule more than accounted for the increased urinary sodium excretion, it was first postulated that the proximal tubule was the sole regula- tor of the natriuresis of ECVE. This postulate was questioned, however, by studies which demonstrated that proximal sodium reabsorption can be depressed without/m ensuing natriuresis, suggesting that a saline load may also inhibit sodium reabsorption in a more distal nephron segment (Howards et al. 1968; Brenner and Berliner 1969; Knox et al. 1973; Knight and Weinman 1977). Evaluation of altered reabsorption during acute ECVE by the "distal" tubule can include the loop of Henle, distal convoluted tubule, and the collecting tubule. The discussion will first focus on evidence for and against the loop of Henle as an important mediator of the natriuresis of volume expansion. On the basis of studies in humans and dogs utilizing free-water clearance as an index of loop of Henle reabsorption, most (Eknoyan et al. 1967; Stein et al. 1967; Leeber et al. 1968; Buckalew et al. 1970b; Factors Influencing Renal Sodium Reabsorption in Volume Expansion 91

Bennett 1973), but not all (Barton et al. 1972), report that ECVE suppresses sodium reabsorption in the loop of Henle. The demonstration that the collecting duct has a marked diluting capacity makes interpretation of free-water clearance data difficult. In contrast, micropuncture studies in which sodium reabsorption along the loop of Henle is estimated by micropuncture of both proximal and distal tubules report increased absolute reabsotption after either isotonic or hypertonic saline infusion in the rat (Giebisch et al. 1964; Cortney et al. 1965; Landwehr et al. 1967; Stein et al. 1973; Kunau et al. 1974)or the dog (Dirks and Seely 1970). Similarly, microperfusion studies, indicate that sodium reabsorption in the loop increased linearly as a function of the load delivered to it, and that ECVE, per se, does not affect its intrinsic reabsorptive capacity (Schnermann 1968; Morgan and Berliner 1969a). In many respects, the transport characteristics of the distal convoluted tubule after volume expansion resemble the loop of Henle. Hayslett, utilizing the sprit-drop technique, reported that reabsorption from the distal convoluted tubule is enhanced by saline infusion (Hayslett et al. 1967). Many free-flow micropuncture experiments have found increased absolute (Kunau et al. 1974; Knox and Gasser 1974; Khuri et al. 1975), and with one exception (Khuri et al. 1975), decreased fractional reabsorption along the distal tubule after volume expansion. In addition, micropuncture experiments in which increased sodium delivery to distal tubules was prevented by reduction of renal perfusion pressure revealed that ECVE had no effect on sodium transport along the superficial distal tubule (Diezi et al. 1980). These results are in agreement with microperfusion studies which found that the rate of sodium reabsorption paralleled sodium delivery to the distal tubule (Morgan and Berliner 1969a). Evidence that ECVE affects sodium transport in the collecting duct has been indirectly evaluated by comparison of sodium delivery in superficial late distal tubules with final urine. Two studies reported diminished fractional sodium reabsorption in the collecting duct after Ringer's infusion when compared with a similar distal delivery of sodium after albumin infusion (Stein et al. 1973;Knox and Gasser 1974). Diezi compared urinary sodium excretion in nondiuretic and volume-expanded rats in which late distal sodium delivery was adjusted to similar levels by means of aortic constriction (Diezi et al. 1980). Urinary sodium excretion was significantly higher in the volume-expanded animals. The comparison of late distal tubular fluid with final urine to estimate collecting duct reabsorption assumes that homogeneity of nephron function exists with regard to sodium handling. If nephron heterogeneity exists, changes in excretion could be due to either alterations in collecting duct reabsorption per se, or could be due to the admixture of fluid from superficial and juxtamedullary nephrons. In order to differentiate between these 92 F.G. Knox and J.A. Haas possibilities, Diezi performed direct micropuncture of the base and tip of the papillary collecting duct. His results showed that after volume expansion, collecting duct fractional sodium reabsorption decreases and absolute sodium reabsorption increases (Diezi et al. 1973). Subsequently, in a similar protocol, Stein evaluated papillary collecting duct reabsorption as well as the possibility of nephron heterogeneity, by comparing the delivery of sodium to the end of the distal tubule superficial nephrons with papillary base delivery (Stein et al. 1976). They found that the delivery of sodium to and reabsorption along the papillary collecting duct were markedly greater during Ringer loading than in hydropenia. Further, the amount of sodium delivered to the papillary base was greater than the delivery to the end of the distal tubule of superficial nephrons during Ringer loading, suggesting that deeper nephrons deliver more sodium to the collecting duct. Finally, the difference in sodium excretion between Ringer loading and hyperoncotic albumin infusion was due to events occurring between the late distal tubule of superficial nephrons and the base of the papillary collecting duct. Although these findings do not exclude the possibility that ECVE may alter collecting duct transport, they concluded that the natriuresis of Ringer loading was due, in part, to increased sodium delivery from inner cortical nephrons, and that the terminal part of the collecting duct reduces the magnitude of the natriuresis. In a subsequent study, direct micropuncture of the deep nephrons during volume expansion confirmed that delivery of sodium to the bend of the loop of Henle in juxtramedullary nephrons was greater than estimates of delivery in the superficial nephrons (Osgood et al. 1978). In addition, micropuncture studies by Reineck showed that the net addition of sodium between the late distal tubule and papillary base seen during Ringer loading is abolished if juxtamedullary nephron function is abolished by drug-induced necrosis (Reineck et al. 1980). With regard to the finding that in the presence of volume expansion, absolute sodium reabsorption along the papillary collecting duct is enhanced, this is in sharp contrast to the conclusions drawn in several microcatheterization studies that either no net reabsorption or even net addition of sodium occurs along the papillary collecting duct during Ringer loading (Sonnenberg 1974, 1975; Wilson and Sonnenberg 1979). The difference between the findings have been ascribed to technique (micropuncture vs microcatheterization) and experimental protocol. In the studies by Sonnenberg, the degree of acute extracellular expansion was markedly greater than in the micropuncture studies and, in addition, urine was reinfused. Urine reinfusion has been shown to be natriuretic (Harris and Yarger 1976). Accordingly, the differences in results between the micropuncture and microcatheterization studies may be due to the urine reinfusion used in the latter, which in turn may depress sodium transport by the papillary collecting duct. Factors Influencing Renal Sodium Reabsorption in Volume Expansion 93

2.4.2 Chronic ECVE Chronic ECVE can be produced experimentally by the administration of mineralocorticoids and large amounts of salt in the diet, followed by a return of sodium excretion to control levels. As mentioned previously, this phenomenon is referred to as "DOCA escape". The nephron sites responsible for escape, and the response of the "escaped" animal to volume expansion have been extensively discussed in a previous paper (Knox et al. 1980). In brief, superimposed volume expansion in the DOCA-escaped rat reveals that fractional reabsorption of sodium is diminished in the superficial nephron segment containing the pars recta and loop of Henle. Fractional reabsorption of sodium is also decreased in deep nephrons proximal to the bend in Henle's loop. Finally, escape occurs from the sodium-retaining effects of mineratocorticoids when sodium delivery from both superficial and deep nephrons is increased enough to overcome sodium reabsorption by the cortical collecting tubule which is stimulated by mineralocorticoids. Chronic ECVE is also a feature of models of heart failure and is characterized by the inability to excrete a saline load. Schneider evaluated proximal sodium reabsorption in dogs with aorta-vena cava (AV) fistulas to produce high output heart failure (Schneider et al. 1971). When these animals are given a saline infusion, the normal inhibition of proximal reabsorption occurs so that delivery from the proximal tubule is markedly increased. However, in these dogs, very little of the increased delivery of sodium appeared in the urine, whereas control dogs had the normal natriuretic response. This observation indicates that sodium transport in some distal nephron segment is altered in response to changes in ECFV. Sturnpe and associates reported similar findings in rats with AV fistulas (Stumpe et al. 1973). Comparing the rats with AV fistulas with normal rats, they found similar delivery of sodium out of the proximal tubule, but enhanced sodium reabsorption along the loop of Henle. In another model of chronic sodium retention, two studies showed that proximal tubular sodium reabsorption was not significantly different in normal animals from animals with thoracic vena cava obstruction after saline loading (Auld et al. 1971 ; Levy 1972). Levy also found enhanced sodium reabsorption in the loop of Henle in the AV fistula group, again suggesting that sodium reabsorption at this nephron segment accounts for the inability of animals with experimental heart failure to excrete a saline load. 94 F.G. Knox and J.A. Haas

2.5 Hormonal Factors

2.5.1 Renin-Ang )tensin-Aldosterone System

It has been prop( ;ed that in the antidiuretic animal, glomerular filtration rate is governed y the orthograde flow of tubule fluid past the macula densa and this in turn may be mediated by the renin-angiotensin system (Schnermann et al. 1970). This hypothesis comes from the observation that in the antidiuretic state, interruption of flow to the macula densa with proximal tubule micropuncture results in a significantly higher single nephron glomerular filtration rate (SNGFR), compared to the SNGFR measured in the distal tubule beyond the macula densa. These experiments imply that distally measured SNGFR represents the true steady-state filtration rate. However, as stated previously, both proximal SNGFR and whole kidney GFR increase in response to extracellular volume expansion (Davidman et al. 1972; Rector et al. 1964), and further, it has been demonstrated that the proximal-distal SNGFR difference is abolished during saline diuresis (Schnermann et al. 1971). If tubuloglomerular feedback participates in the regulation of filtration rate, one would expect during volume expansion, when distal sodium chloride delivery is elevated, a decrease rather than an increase in filtration rate. Dev and co-workers postulated that this apparent discrepancy might be due to variations of sensitivity of the feedback system as a function of salt intake (Dev et al. 1974). They found that in rats with a sodium intake of greater than 6 mEq/day, the feedback sensitivity (i.e., proximal-distal SNGFR difference) was abolished. In an extension of these studies, Schnermann et al. measured feedback sensitivity by comparing proximal flow rate, filtration rate, and stop-flow pressure in response to loop of Henle perfusion in control rats and DOCA-saline treated rats (Schner- mann et al. 1975). The DOCA-saline treated rats exhibited a blunted feedback responsiveness compared to controls, accompanied by a significant decrease in juxtaglomerular renin activity. From these observations it was postulated that in animals, either acutely or chronically volume expanded, feedback response is blunted. This impaired feedback accounts for the increase in SNGFR and whole kidney GFR which accompanies ECVE. However, a subsequent study concluded that autoregulation of whole kidney and proximal filtration rate occurred in both sodium depleted and expanded animals in which there was a threefold to four- fold difference in measured renal renin content (Marchand 1978). Studies by de Wardener et al. demonstrated that acute ECVE increased sodium excretion depsite the administration of large doses of 9 a-fluorohydrocortisone and reductions in GFR (De wardener et al. 1961). These experiments were taken as evidence that increases in sodium excretion Factors Influencing Renal Sodium Reabsorption in Volume Expansion 95 following acute volume expansion were independent of changes in adrenocortical hormone secretion. However, these experiments do not rule out the possibility that acute reductions of aldosterone secretion during volume expansion may be a contributing mechanism for the natriuresis. This possibility was studied by comparing the natriuresis of volume expansion in the intact and adrenalectomized rat. One difficulty of evaluating renal function in the absence of mineralocortocoids is that this maneuver is achieved by adrenalectomy, which usually lowers blood pressure and results in a decreased GFR. Even so, it has been demonstrated that volume natriuresis in response to either isotonic saline or blood volume expansion is similar in intact and adrenalectomized rats (Cortney 1969; Veress and Pearce 1974). Evaluating the effect of variations in endogenous secretion ofmineralo- corticoids on the natriuresis of ECVE is difficult. In order to achieve a stimulation of aldosterone secretion, animals are given a low sodium diet and/or administered a diuretic. These maneuvers also contract the ECFV, which in itself affects the renal response to ECVE (Higgins 1971). Given the fact that animals administered exogenous mineralocorticoids acutely respond with a diuresis of the same magnitude as untreated ones makes it unlikely that suppression of aldosterone secretion is the primary cause of the saline diuresis. When the ECFV is gradually increased by the chronic administration of mineralocorticoids, an initial retention of sodium is followed by a return of sodium excretion to control levels. This phenomenon is referred to as escape from the sodium retaining effects of mineralocorticoids (Knox et al. 1980). The importance of the state of sodium balance in determining the natriuretic response to saline loading has been discussed in Sect. 2.3.5). As mentioned before, an augmented diuretic response to ECVE has been demonstrated in DOCA-escaped dogs. This phenomenon has also been reported in normal humans given 9 ~-fluorohydrocortisone (Strauss and Earley 1959) and in patients with primary aldosteronism (Biglieri and Forsharn 1961). In normal subjects, infusion of isotonic saline suppresses aldosterone secretion, whereas patients with primary aldosteronism fail to do so (Espiner et al. 1967). However, both respond with marked natriuresis. This responsiveness of sodium excretion to ECVE reinforces the independence from aldosterone secretion.

2.5. 2 Natriuretic Hormone

Several studies have presented evidence suggesting that there is a natriuretic hormone which contributes to the increase in urinary sodium excretion in response to changes in ECFV expansion. Attempts to prove the existence of a natriuretic hormone have employed several different techniques. The first, cross-circulation, was initially used by de Wardener 96 F.G. Knox and J.A. Haas et al. (t 961 ). Cross-circulation involves joining the circulation of two animals via a shunt. One animal serves as a donor and has its ECFV acutely or chronically expanded. In the second animal (recipient) ECFV is kept constant and urinary excretion of sodium is monitored. These cross-circulation studies induce an increase in urinary sodium excretion in the assay kidney, which is taken as evidence of a change in the circulating concentration of a natriuretic hormone. Expansion of the donor animal with saline has been performed by several groups (de Wardener et al. 1961 ; Johnston and Davis 1966; Johnston et al. 1967; Blythe et al. 1971 ; McDonald et al. 1967). A number of similar experiments have been performed, except, instead of using saline, the donor animals has been expanded with blood or iso-oncotic albumin and the assay kidney is in situ (Bahlrnann et al. 1967; Pearce et al. 1969) or is isolated and perfused (Lichardus and Pearce 1966; KaIoyanides and Azer 1971;KaIoya- nides et al. 1977). The cross-circulation experiments with saline have been criticized due to the dilution of the blood in the recipient animal (hematocrit, protein concentration), which in itself could result in a natriuresis in the recipient animal. Two groups have addressed this problem in experiments in which they expanded the donor animal with equilibrated blood from a reservoir (Knox et al. 1968;Bengele et al. 1972). In these animals, as well as those expanded with saline, the sodium excretion in the recipient is usually only a small fraction of that in the donor. Another approach to detect a natriuretic hormone involves obtaining extracts of plasma, urine or tissue from volume-expanded donors and comparing the natriuresis when injected into bioassay rats with extracts from nonexpanded donors (Sealey et al. 1969; Viskoper et al. 1971; Brown et al. 1972; Gonick and Saldanha 1975). Again, the ensuing natriuresis is usually modest and sometimes accompanied by an increase in GFR and/or renal blood flow (RBF). These findings raise questions regarding the specificity of the extract, as well as the physiologic significance. In vitro assays for natriuretic hormone have involved the measurements of ion transport by the isolated frog skin or toad bladder, the transport of p-aminohippurate by kidney slices, the activity of kidney ATPase and transport of sodium and potassium by renal tubule fragments (Bricker et al. 1968;Buckalew et al. 1970a; Clarkson et al. 1970;Nutbourne et al. 1970; Katz and Genant 1971; Gruber et al. 1980). Many of the findings with in vitro assays have not been confirmed in other laboratories, and the question arises whether the results are relevant to a natriuresis in vivo. The evidence that the natriuresis of ECVE is due, in part, to a natriuretic hormone remains both equivocal and enticing. Factors Influencing Renal Sodium Reabsorption in Volume Expansion 97

2.5.3 Prostaglandins

Saline loading results in an increase in the urinary excretion of PGA and PGE in humans (Papanicolaou et al. 1975), as well as significant changes in intrarenal PGA2 concentration in the rabbit (Attallah and Lee 1973). Following inhibition of PG synthesis by indomethacin administration, sodium excretion in chronically saline loaded rabbits decreased accompanied by a significant fall of PGA2 concentration in the outer medulla and papilla. Inhibition of prostaglandin synthesis by either fatty acid deprivation (Rosenthal et al. 1974), aspirin (Susic and Sparks 1975), or indomethacin (Dusing et al. 1977b) has been shown to reduce the natriuresis of saline loading. Dusing and coworkers studied the effect of prostaglandin synthesis inhibition in ECV-expanded rats on total renal plasma flow and its intrarenal distribution as well as on GFR (Dusing et al. 1977a). In ECV-expanded rats, indomethacin had no effect on GFR, but total renal plasma flow was significantly decreased, which was due to a decrease in outer cortical blood flow. To assess the specific sites of enhanced chloride reabsorption in ECV-expanded rats treated with either indomethacin or meclofenamate,Higashihara performed free flow micropuncture experiments in the Munich-Wistar rats (Higashira et al. 1979). The results showed that inhibitors of prostaglandin synthesis increase chloride transport in the thick ascending loop of Henle, and/or the cortical and outer medullary collecting tubule. Several studies, however, have reported results which showed that inhibition of prostaglandin synthesis results in a significant natriuresis after saline infusion in rabbit, dog, and man (Oliw et al. 1978; Kirschenbium and Stein 1976; Mountokolakis et al. 1978). In view of this controversy, the physiologic significance of prostaglandins in mediating the natriuresis of volume expansion is, therefore, not settled.

2.5.4 Antidiuretic Hormone

The possibility that the natriuresis of ECVE is due to a dilution of antidiuretic hormone, thus leading to a diuresis, has been eliminated. Exo- genous administration of vasopressin to assure a maximal reabsorption of water has little or no effect on the natriuresis of ECVE (de 14/ardener et al. 1961 ; Earley and Friedler 1965a). Further, acute hypophysectomy does not significantly alter the natriuretic response to volume expansion (Kaloyanides et al. 1977).

2.5.5 Parathyroid and Thyroid Hormone

Extracellular volume expansion with calcium-free Ringer's solution results in increases in parathyroid hormone (PTH) secretion as a result of a fall 98 F.G. Knox and J.A. Haas in plasma ionized calcium concentration (Schneider et al. 1975). Micro- puncture studies in dogs have shown that infusion of parathyroid hormone results in decreased sodium and phosphate reabsorption by the proximal tubule (Agus et al. 1971). From these observations one could hypothesize that PTH may play an important role in the natriuresis accompanying ECVE. However, it has been demonstrated that while parathyroid hormone significantly affects the phosphaturia of ECVE, the natriuresis is not dependent on the presence of PTH (Schneider et al. 1975). Holmes and DiScala have shown that hypertonic saline infusion in hypothyroid rats resulted in fractional excretion of 45% of the filtered load, whereas no control animal excreted more than 12% (Holmes and DiScala 1970). This suggests that a thyroid hormone provides a basal support for sodium transport during ECVE.

2.5.6 Neurohumoral

It has been demonstrated that the natriuresis of blood volume expansion is virtually eliminated by high spinal cord section in the dog (Pearce and Sonnenberg 1965). Gilmore and Daggett observed a blunted natriuresis to volume expansion with 6% dextran in isotonic saline following chronic cardiac denervation (Gilmore and Daggett 1966). This was interpreted as being due to interruption of nonvagal afferent pathways from volume receptors near the heart. Knox et al. confirmed that cardiac denervation attenuates the natriuresis of saline loading, but found that proximal reabsorption was depressed to a similar degree in response to ECVE as occurs in animals with cardiac innervation (Knox et al. 1967). Subsequently, McDonald and co-workers investigated the influence of afferent and efferent neural pathways on the natriuresis of ECVE (McDonald et al. 1970). High spinal section, which interrupts both afferent and efferent pathways, markedly reduced the natriuretic response to volume expansion. However, cord transection at the T6 level, which preserved thoracic but not abdominal innervation, failed to blunt the natriuretic response. Further, interruption of afferent neural pathways by sectioning nerve roots from C8 to T6, did not impair the natriuresis of ECVE. In this same group of animals, vagotomy also failed to affect the natriuretic response. Similar findings in regard to vagotomy have been shown in response to blood volume expansion (Pearce and Lichardus 1967). These results strongly suggest that the adrenergic efferent rather than afferent pathways are important in the natriuresis associated with volume expansion in dogs. Studies have demonstrated that renal denervation in the volume expanded animals leads to an. additive natriuresis (Bengele et al. 1972; Bello-Reuss et al. 1977), which was due to a further decrease in proximal reabsorption (Bello-Reuss et al. 1977). Further, low frequency electrical Factors Influencing Renal Sodium Reabsorption in Volume Expansion 99 stimulation of the denervated rat kidney reverses the increase in sodium excretion seen in saline expansion (Bello-Reuss et al. 1976). These results are consistent with the notion that a tonic nerve influence on the kidney persists during volume expansion. Studies in the rabbit have, in fact, demonstrated decreased efferent neural traffic during blood volume expansion (Clement et al. 1972). Previous studies have assessed the interrelationship between the cate- cholaminergic system and ECFV expansion with adrenergic blockade, or measurement of catecholamine blood levels and urinary excretion during acute volume expansion. Gill observed an enhanced natriuresis to saline infusion following guanethidine administration (Gill et al. 1964). Alexan- der observed that sympathetic nerve activity, as shown by measurement of plasma dopamine ~-hydrooxylase and urinary norepinephrine, is suppressed during saline infusion (Alexander et al. 1974). Additional studies have confirmed that plasma levels of norepinephrine and epinephrine are reduced during acute saline loading in dogs (Faucheux et al. 1977; Carriere et al. 1978). These reductions were greater than could be accounted for by hemodilution alone. These data indicate that there may be a direct effect of volume expansion to lower circulating plasma catecholamines, and thereby reduce renal sympathetic tone during volume expansion.

2.6 Response of the Perinatal Kidney to Volume Expansion

When a perinatal mammal is given an ECVE, the result is a smaller absolute and fractional excretion of sodium compared to an adult receiving an equivalent sodium load. This response has been described in several different species (Aperia et al. 1972;Merlet-Benichou and de Rouffignac 1977; Hurley et al. 1977; Bengele and Solomon 1974; Kleinman and Reuter 1974) and the reasons underlying the phenomenon have been ascribed to several different factors. Among these are rate of glomerular filtration, intrarenal distribution of blood flow, physical and hormonal factors, and inability of the proximal and/or distal tubule to decrease sodium reabsorption in response to a saline load. The attenuated natriuretic response to volume expansion in the neonate was initially attributed to a relatively small increase in glomerular filtration rate in association with volume expansion. Additionally, a change in the intrarenal distribution of blood flow was postulated to influence sodium handling. The results of studies in newborn and adult dogs, however, show that GFR increases similarly in response to volume expansion in dogs of all ages, and that there is no correlation between the blunted natriuresis and the change in intrarenal blood flow distribution in the newborn dog (Kleinman and Reuter 1974, Goldsmith et al. 1979). 100 F.G. Knox and J.A. Haas

The relationship between changes in physical factors and proximal reabsorption after volume expansion in the neonate is unknown. It has been shown that the oncotic pressure of young rats is lower than that of mature rats (Allison et al. 1972). This observation raises the possibility that in the neonate, volume expansion may not decrease peritubular oncotic pressure to the same extent as the mature animal. However, mor- phological studies demonstrate widening of lateral intercellular spaces during volume expansion in the neonate rat, quantitatively the same as in the mature rat (Misanko et al. 1979). Another possible explanation for the age-related differences in the natriuretic response to volume expansion is elevated blood levels of aldosterone present in the neonate (Beitens et al. 1972). It is possible that the neonate kidney is exposed to a greater salt-retaining stimulus due to the amount of aldosterone reaching the kidney, although there is a normal response to volume expansion in the mature animal pretreated with exogenous mineralocorticoids. It has been demonstrated that high sodium intake prior to volume expansion enhances the natriuretic response in puppies (Steichen and Kleinman 1975). Results of experiments in newborn dogs demonstrate that the natriuretic response to volume expansion can be restored to adult levels by the administration of oxytocin. Furthermore, the effect was localized to a nephron segment beyond the proximal tubule (Kleinrnan and Banks 1980). Two studies in newborn dogs were carried out to determine whether the attenuated natriuresis to volume expansion is due to enhanced proximal or distal nephron sodium reabsorption. The protocol involved blocking sodium reabsorption beyond the proximal tubule by administration of diuretics, with the rationale being that a change in sodium reabsorption occurring during volume expansion could be attributed to the proximal tubule (Kleinrnan 1975; Banks and Kleinman 1978). When distal tubular sodium reabsorption was blocked by administration of either amiloride or chlorothiazide and ethacrynic acid, saline expansion increased fractional sodium excretion in the neonate to the same degree as in the untreated volume-expanded adult. These results suggest that the proximal tubule of adult and neonatal dogs respond similarly to volume expansion by decreasing proximal reabsorption, but that sodium excretion in more distal segments of the nephron is augmented in the neonate in response to volume expansion. Factors Influencing Renal Sodium Reabsorption in Volume Expansion 101

2.7 Gastrointestinal and/or Hepatoportal Control of Renal Sodium Excretion

The hypothesis that a sodium ion receptor exists in the gut or portal circulation came from experiments which showed a greater natriuresis when a hypertonic sodium chloride load was given orally rather than intraven- ously. Lennane and Carey found that in both rabbit and man, oral sodium loading is followed by a greater natriuresis than intravenous sodium loading (Lennane et al. 1975a,b; Carey et al. 1976; Carey 1978). Because there were no significant changes in either urinary or plasma aldosterone levels between the oral and intravenous groups, it was suggested that the gastrointestinal tract influences renal sodium excretion by a mechanism independent of aldosterone. However, other investigators have been unable to confirm the hypothesis that a sodium receptor within the GI tract or portal circulation is important in regulating sodium excretion. Hanson found no difference in sodium excretion in dogs receiving hypertonic sodium chloride via the GI tract vs an intravenous infusion (Hanson et al. 1980). Similarly, results of experiments in rabbits could show no difference in urinary sodium excretion following either a gastrointestinal or intravenous administration of hypertonic sodium chloride (Obika et al. 1981). Variations in food intake between the studies may account for the differences seen in dog, rabbit, and man. It has also been reported that the natriuretic response to a saline load is greater when the infusion is administered into the portal vein rather than into a systemic vein, suggesting that there are osmoreceptors within the portal or hepatic circulation controlling the natriuresis following oral sodium chloride loading. Passo reported that hypertonic infusions of sodium chloride into the hepatic portal vein of the cat results in a greater increase in sodium excretion than do comparable femoral vein infusions (Passo et al. 1972). Similar results were observed in the dog (Daly et al. 1967; Strandhoy and Williamson 1970). However, using similar proto- cols, several studies have been unable to demonstrate an enhanced natriuresis following hypertonic sodium chloride infusions into the portal vein of the dog (Schneider et al. 1970; Potkay and Gilmore 1970; Kap- teina et al. 1978). It is difficult to reconcile the differences reported in the above studies; therefore, experimental evidence for a GI or portal sodium receptor is not conclusive. Evidence has been presented that the liver may play an important role in renal tubular sodium reabsorption. Enhanced renal sodium reabsorption and impaired renal concentrating ability may be associated with the heptatorenal syndrome (Shear et al. 1965). Liver damage produced by ligation of the common bile duct has been used as an experimental model 102 F.G. Knox and J.A. Haas for the hepatorenal syndrome (Mullane and Gliedman 1970). It has been reported that dogs with ligation of the CBD have a blunted natriuresis during ECVE, which was attributed to an enhanced proximal and distal tubular sodium reabsorption (Better and Massry 1972). In an ensuing study, it was noted that ECVE does not elicit a renal vasodilation in animals with CBD ligation, and it was speculated that this may be partly responsible for the blunted natriuresis (Melman and Massry 1977).

3 Summary

This review summarizes factors influencing renal sodium reabsorption in volume expansion. The influence of renal blood flow and intrarenal blood flow distribution on renal sodium excretion during extracellular volume expansion is unclear. There is no convincing experimental evidence that an increase in renal blood flow per se or a change in the intrarenal distribution of renal blood flow is responsible for the natriuresis of ECVE. An increase in whole kidney glomerular filtration rate has been ruled out as the primary cause of the increase in sodium excretion following volume expansion, and several studies have shown that ECVE is not associated with a change in the intrarenal distribution of GFR. Several investigators propose that the primary variables regulating sodium transport during ECVE are physical factors in the peritubule circulation. There is much evidence to indicate that acute ECVE decreases sodium reabsorptibn by the proximal tubule. In animals with chronic ECVE, sodium transport along the loop of Henle is decreased. The role of hormonal factors in mediating the natriuresis of volume expansion is controversial. Although current evidence suggests that neurohumoral factors may be important, a role for other hormones, including "natriuretic hormone", is still not settled. In addition, there is no convincing experimental evidence to date to indicate that a sodium ion receptor exists in the gut or portal circulation.

Acknowledgments. Studies from our laboratory were supported by NIH grant HL 14133 and the Mayo Foundation. The authors wish to thank June Hanke for her secretarial assistance. Factors Influencing Renal Sodium Reabsorption in Volume Expansion 103

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Migdal S, Alexander EA, Bruns F J, Riley AL, Levinsky NG (1975) Effect of hemodilution on the distribution of renal blood flow. Circ Res 36:71-75 Misanko BS, Bengele HH, Solomon S (1979) Renal response to Ringer expansion in developing rats. Biol Neonate 35: 52-59 Morgan T, Berliner RW (1969a) A study by continuous microperfusion of water and electrolyte movements in the loop of Henle and distal tubule of the rat. Nephron 6:388-405 Morgan T, Berliner RW (1969b) In vivo perfusion of proximal tubule of the rat: Glo- merular balance. Am J Physiol 217:992-997 Mountokalakis T, Karambasis T, Mayopoulou-Symvoulidou MG, Merikas G (1978) Effect of inhibition of prostaglandin synthesis on the natriuresis induced by saline infusion in man. Clin Sci Mol Med 54:47-50 Mullane JF, Gliedman ML (1970) Renal response to saline load in experimental liver disease. J Surg Res 10:519-523 Munck O, de Bono E, Mills IH (1970) Distribution of blood flow in the renal cortex during saline loading. Clin S ci 38:699-712 Nissen OI (1968) Changes in the filtration fractions in the superficial and deep venous drainage area of the cat kidney due to fluid loading. Acta Physiol Scand 73:320 328 Nutbourne DM, Howse JD, Schrier RW, Talner LB, Ventom MG, Verroust PJ, de War- dener HE (1970) Effect of expanding the blood volume of a dog on the short cir- cuit current across the isolated frog skin incorporated in the dog's circulation. Clin Sci 38:33-34 Obika LFO, Fitzgerald EM, Gleason SD, Zucker A, Schneider EG (1981) Lack of evidence for gastrointestinal control of sodium excretion in unanesthetized rabbits. Am J Physiol 240:F94-F100 Oliw E, Kover G, Larsson C, Anggard E (1978) Indomethacin and diclofenac increase sodium and water excretion after extracellular volume expansion in the rabbit. Eur J Pharmacol 49:381-388 Osgood RW, Lameire NH, Sorkin MI, Stein JH (1977) Effect of aortic clamping on proximal reabsorption and sodium excretion in the rat. Am J Physiol 232:F92- F96 Osgood RW, Reineck H J, Stein JH (1978) Further studies on segmental sodium transport in th6 rat kidney during expansion of the extracellular fluid volume. J Clin Invest 62:311-320 Ott CE, Haas JA, Cuche J-L, Knox FG (1975) Effect of increased peritubule protein concentration on proximal tubule reabsorption in the presence and absence of extracellular volume expansion. J Clin Invest 55: 612-620 Papanicolaou N, Safar M, Hornych A, Fontaliran F, Weiss Y, Bariety J, Milliez P (1975) The release of renal prostaglandins during saline infusion in normal and hypertensive subjects. Clin Sci Mol Med 49:459-463 Papper S, Belsky JL, Bleifer KH (1960) The response to the administration of isotonic sodium chloride-lactate solution in patients with essential hypertension. J Clin Invest 39:876-884 Passo SS, Thornborough JR, Rothboller AB (1972) Hepatic receptors in control of sodium excretion in anesthetized cats. Am J Physiol 224:373-375 Pearce JW, Lichardus B (1967) Effects of vagotomy and renal denervation on renal response to blood volume expansion. Can J Physiol Pharmacol 45:689-703 Pearce JW, Sonnenberg H (1965) Effects of spinal section and renal denervation on the renal response to blood volume expansion. Can J Physiol Pharmacol 43:211 - 224 Pearce JW, Sonnenberg H, Veress AT, Ackerman U (1969) Evidence for a humoral factor modifying the renal response to blood volume expansion in the rat. Can J Physiol Pharmacol 47: 377-386 Potkay S, Gilmore JP (1970) Renal response to vena caval and portal venous infusions of sodium chloride in unanesthetized dogs. Clin Sci Mol Med 39:13-20 Factors Influencing Renal Sodium Reabsorption in Volume Expansion 111

Poujeol R, Bonvalet JP, de Rouffignac C (1975) Glomerular filtration rate and microsphere distributions in single nephron of rat kidney. Pfliigers Arch 357:291-301 Quinn MD, Marsh DJ (1979) Peritubular papillary control of proximal tubule reabsorption in the rat. Am J Physiol 236:F478-F487 Rector FC Jr, Giesen GV, Kiil F, Seldin DW (1964) Influence of expansion of extracellular volume on tubular reabsorption of sodium independent of changes in glomerular filtration rate and aldosterone activity. J Clin Invest 43:341-348 Rector FC Jr, Sellman JC, Martinez-Maldonado M, Seldin DW (1967) The mechanism of suppression of proximal tubular reabsorption by saline infusions. J Clin Invest 46:47-56 Reineck H J, Parma R, Barnes JL, Osgood RW (I 980) Nephron heterogeneity in renal excretion of sodium and potassium in the rat. Am J Physiol 239:F187-F193 Richet G, Hornych A (1969) The effect of an expansion of extracellular fluids on net Na flux in the jejunum of rats. Nephron 6:365-378 Rosenthal J, Simone PG, Silbergleit A (1974) Effects of prostaglandin deficiency on natriuresis, diuresis and blood pressure. Prostaglandins 5:435-440 Rouffignac C de, Bonvalet JP (1970) Variations in GFR of single superficial and deep nephrons under various conditions of sodium intake in the rat. Pfliigers Arch 317: 141-156 Rumrich G, Ullrich KJ (1968) The minimum requirements for the maintenance of sodium chloride reabsorption in the proximal convolution of the mammalian kidney. J Physiol (London) 197:69-70 Sato K (1975) Reevaluation of micropuncture techniques: Some of the factors which affect the rate of fluid absorption by the proximal tubule. In: Angielski S, Dubach UC (eds) Current problems in clinical biochemistry; biochemical aspects of renal function, vol 4, pp 175-187 Schneider EG, Dresser TP, Lynch RE, Knox FG (1971) Sodium reabsorption by proximal tubule of dogs with experimental heart failure. Am J Physiol 220:952-957 Schneider EG, Goldsmith RS, Arnaud CD, Knox FG (1975) Role of parathyroid hormone in the phosphaturia of extracellular fluid volume expansion. Kidney Int 7: 317-324 Schneider EG, Lynch RE, Willis LR, Knox FG (1972) Single-nephron filtration rate in the dog. Am J Physiol 222:938-944 Schneider EG, Davis JO, Robb CA, Baumber JS, Johnson JA, Wright FS (1970) Lack of evidence for a hepatic osmoreceptor mechanism in conscious dogs. Am J Physiol 218:42-45 Schnermann J (1968) Microperfusion study of single short loops of Henle in rat kidney. Pfliigers Arch 300:255-282 Schnerman J, Hermle M, Schmidmeier E, Dalheim H (1975) Impaired potency for feedback regulation of glomerular filtration rate in DOCA escaped rats. Pfliigers Arch 358:325-338 Schnermann J, Davis JM, Wunderlich P, Levine DZ, Horster M (1971) Technical problems in the micropuncture determination of nephron filtration rate and their functional implications. Pfliigers Arch 329:307-320 Schnermann J, Wright FS, Davis JM, v Stackelberg W, Grill G (1970) Regulation of superficial nephron filtration rate by tubulo-glomerular feedback. Pfliigers Arch 318:147-175 Schrier RW, Earley LE (1970) Effects of hematocrit on renal hemodynamics and sodium excretion in hydropenic and volume-expanded dogs. J Clin Invest 49: 1656-1667 Schrier RW, Fein RL, McNeil JS, Cirksena WJ (1969) Influence of interstitial fluid volume expansion and plasma sodium concentration on the natriuretic response to volume expansion. Clin Sci 36:371-385 Schrier RW, McDonald KM, Marshall RA, Lauler DP (1968) Absence of natriuretic response to acute hypotonic intravascular volume expansion in dogs. Clin Sci 34: 57-72 112 F.G. Knox and J.A. Haas

Schrier RW, McDonald KM, Wells RE, Lauler DP (1970) Influence of hematrocrit and colloid on whole blood viscosity during volume expansion. Am J Physiol 218: 346-352 Sealey JE, Kirshman D, Laragh JH (1969) Natriuretic activity in plasma and urine of salt-loaded man and sheep. J Clin Invest 48:2210-2224 Shear L, Kleinerman J, Gabuzda GJ (1965) Renal failure in patients with cirrhosis of the liver. I. Clinical and pathologic characteristics. Am J Med 39:184-198 Sonnenberg H (1974) Medullary collecting duct function in anti-diuretic and in salt- or water-diuretic rats, Am J Physiol 501-506 Sonnenberg H (1975) Secretion of salt and water into the medullary collecting duct of Ringer-infused rats. Am J Physiol 228:565-568 Sonnenberg H, Solomon S (1969) Mechanism of natriuresis following intravascular and extracellular volume expansion. Can J Physiol Pharmacol 47:153-159 Spitzer A, Windhager EE (1970) Effect of peritubular oncotic pressure changes on proximal tubular fluid reabsorption. Am J Physiol 218:1188-1193 Steichen J J, Kleinmann LI (1975) Influence of dietary sodium intake on renal maturation in unanesthetized canine puppies. Proc Soc Exptl Biol Med 148:748-751 Stein JH, Osgood RW, Ferris TF (1972) Effect of volume expansion on distribution of glomerular filtrate and renal cortical blood flow in the dog. Am J Physiol 223: 984-990 Stein JH, Osgood RA, Kunau RT Jr (1976) Direct measurement of papillary collecting duct sodium transport in the rat. J Clin Invest 58:767-773 Stein JH, Osgood RW, Boonjaren S, Ferris T (1973) A comparison of the segmental analysis of sodium reabsorption during Ringers and hyperoncotic albumin infusion in the rat. J Clin Invest 52:2312-2323 Stein RM, Abramson RG, Kahn T, Levitt MF (1967) Evidence for a saline-induced limit on distal tubular sodium transport. J Clin Invest 46:1205-1214 Strandhoy JW, Williamson HE (1970) Evidence for an hepatic role in the control of sodium excretion. Proc Soc Exptl Biol Med 133:419-422 Strauss MB, Eartey LE (1959) An inquiry into the role of "sodium-retaining" steroids in the homeostasis of body sodium in man. Trans Assoc Am Physicians 72: 200 -206 Stumpe KO, Lowitz HD, Ochwadt B (1970) Fluid reabsorption in Henle's loop and urinary excretion of sodium and water in normal rats and rats with hypertension. J Clin Invest 49:1200-1212 Stumpe KO, Solle H, Klein H, Kruck F (1973) Mechanism of sodium and water retention in rats with experimental heart failure. Kidney Int 4:309-317 Susic D, Sparks JC (1975) Effects of aspirin on renal sodium excretion, blood pressure, and plasma and extracellular fluid volume in salt loaded rats. Prostaglandins 10:825-831 Tucker B J, Blantz RC (1978) Determinants of proximal tubular reabsorption as mechanisms of glomerulotubular balance. Am J Physiol 235:F 142-F 150 Velasquez MT, Notargiacomo MV, Cohn JN (1973) Comparative effects of saline and mannitol on renal cortical blood flow and volume in the dog. Am J Physiol 224: 322-327 Velasquez MT, Notargiacomo MV, Cohn JN (1975) Segmental changes in intrarenal hemodynamics during saline infution in the dog. Am J Physiol 228:1663-1668 Veress AT, Pearce JW (1974) The effect of acute adrenalectomy on volume natriuresis in the rat. Proc Soc Explt Biol 145:533-536 Viskoper JR, Czaczkes JW, Schwartz N, Ullmann TD (1971) Natriuretic activity of a substance isolated from human urine during the excretion of a salt load. Nephron 8:540-548 Wallin JD, Blantz RC, Katz MA, Andreucci VE, Rector FC Jr, Seldin DW (1971) Effect of saline diuresis on intrarenal blood flow in the rat. Am J Physiol 221: 1297-1304 Factors Influencing Renal Sodium Reabsorption in Volume Expansion 113

Wardener HE de, Mills IH, Clapham WF, Hayter CJ (1961) Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 21:249-258 Wilson DR, Sonnenberg H (1979) Medullary collecting duct function in the remnant kidney before and after volume expansion. Kidney Int 15:487 -501 Windhager EE, Lewy JE, Spitzer A (1969) Intrarenal control of proximal tubular reabsorption of sodium and water. Nephron 6:247-259 Rev. Physiol. Biochem. Pharmacol., Vol. 92 ©by Springer-Verlag 1982

Hormonal Coordination of the Immune Response

JEAN COMSA, HUBERT LEONHARDT, and HARTMUT WEKERLE *

Contents 1 The Thymus ...... 1 16 1.1 Immunological Consequences of Thymectomy ...... 117 1.2 Cell-to-Cell Interaction in the Immune Response ...... 118 1.3 Role of the Thymus in Immunotolerance ...... 119 1.4 Role of the Thymus in Cytotoxic and Graft Immune Reactions .... 120 1.5 Suppressor T Cells ...... 121 1.6 Mechanism of the T-Cell-B-Cell Interaction ...... 122 1.7 Distinction of T and B Cells ...... 125 1.8 Subpopulations of T Ceils ...... 126 1.9 Role of the Thymus in Organization and Generation of Immune Response ...... 127 1.9.1 Immunological Functions of T Lymphocytes ...... 128 1.9.2 T Lymphocyte Differentiation Markers ...... 129 1.9.3 Role of T Lymphocyte Sublines in the Organization of the Immune System ...... 130 1.9.4 Generation of Antigen Diversity in the Thymus ...... 132 1.10 Substitutive Therapy for the Thymoprivic Condition ...... 133 1.10.1 Thymic Grafts ...... 133 1.10.2 Transfusion of Thymic or T Cells ...... 134 1.10.3 Cell-Free Thymic Preparations ...... 134 1.10.4 Thymic Extracts ...... 135

2 The AdrenalCortex ...... 145

3 The Thyroid ...... 150

4 The Gonads ...... 151

5 The AdenohypophyNs ...... 153

Influence of Hypophyseal Hormones on the Immune Response ...... 154 6.1 Growth Hormone ...... 154 6.2 Corticotropin ...... 155

Mechanisms of Hormonal Influence on the Immune Response ...... 156 7.1 Cellular and Subcellular Aspects of Hormonal Action ...... 156 7.1.1 The Corticosteroids ...... 156 7.1.2 Peptidic Hormones ...... 158 7.1.3 Cyclic Adenosine Monophosphate (cAMP) ...... 158 7.1.4 Synthetic Polyribonucleotides ...... 158

* Universit~t des Saarlandes, Medizinische Fakultfit, D-665 Homburg (Saar) 116 J. Comsa et al.

7.2 Hormonal Interactions ...... 159 7.2.1 Consequences of Thymectomy on the Endocrines ...... 159 7.2.2 Thymus-Thyroid ...... 161 7.2.3 Thymus-Adrenal ...... 161 7.2.4 Thymus-Adenohypophysis ...... 162 8 Possible Alimentary Influences ...... 165 9 General Conclusions ...... 166

References ...... 168

For the last 20 years, hormonal influences on the immune system have been subject to an immense amount of research. The documentation accumulated thus far obviously differs from one endocrine organ to another and in some cases it has been one-sided. About 10 000 publica- tions have been dedicated to the role of the thymus in immunology, whereas references to the role of the thyroid or the gonads are scarce. Immunological work on the adrenal cortex has been considerable, but (with few exceptions) has dealt only with the immunosuppressive influence of corticoids administered in "unphysiological" amounts to intact animals. The concept of a proper hormonal regulation of the immune response, i.e., the role of interactions of several endocrines, under a possible ultimate neurohormonal control, emerged only recently, and its elucidation is largely a matter for future research. The subject of the present review is, among other things, an attempt to indicate possible directions for this research. Thus, the present review has to record immunological and endocrinological documentation. Since a result of these considerations is that the proper endocrinological point of view has for some reason been neglected so far by immunologists (except, of course, for the thymic hormones), this point will be emphasized below. Immu- nological documentation will be summarized and limited to information necessary to understand present knowledge in endocrinology.

1 The Thymus

As stated above, more than 10 000 references may be found concerning the role of the thymus in immunity [see reviews 216, 218,255,329, and 504]. From this tremendous bibliography some valid conclusions can be drawn. A chronological review presents a good idea of the course of this knowledge. Hormonal Coordination of the Immune Response 117

1.1 Immunological Consequences of Thymectomy

The thymus of immunized rabbits contains no antibodies [62] and the thymus of immunized guinea pigs yields no antibodies in the incubation medium [191. However, thymectomy is followed by a severe decrease in antibody production in weanling guinea pigs injected with Salmonella antigens [115, 2681, rats [e.g. 24, 5251, rabbits [12, 35], and mice [e.g. 15,252,256,261,362, 469,493, 557] suffer from a severe immunodeficiency when thymectomized at birth. This is not constant [e.g. 271,252, 35, 484], but thymectomy in prepuberal mice results in immunodefic- ciency within a certain delay. The accelerated development of experimental tumors, whether induced by polyoma virus [133, 315], by synthetic carcinogens [368] or by grafting malignant cells [e.g. 421 ], was explained as an immune deficiency. The rejection of allografts is delayed in mice and rats thymectomized at birth [e.g. 14, 41,348, 362, 3711, while in weanling rats, the rejection of skin allografts is only slightly delayed [ 123]. The immune deficiency was considered to be the reason for the characteristic wasting of thymectomized mice [e.g. 24] since axenic thymectomized mice do not waste [e.g. 543], and this was generally confirmed [410]. This immunodeficiency of thymectomized animals may be paralleled with the abnormal composition of their immunoglobulins. In these animals, IgM is normal, IgG is deficient, and IgA is absent. Furthermore, sensitized, thymectomized animals fail to perform the switch from IgM production to IgG production during the immune response [see 67 for review]. The immunodeficiency of thymectomized animals (mostly mice) was confirmed by a large and still increasing number of techniques: 1. The plaque-forming cell assay [273]. Plaque-forming cells are severely reduced in number in the spleen of thymectomized mice injected with sheep erythrocytes [e.g. 196, 371]. 2. The rosette-forming cell test [e.g. 57]. Rosette-forming cells (with sheep erythrocytes) decreased in number in the spleen of neonatally thymectomized mice [e.g. 29]. 3. The graft-vs-host reaction in its in vivo [57, 468] and in vitro form [22]. Spleen cells from thymectomized donors were found to be unable to induce the in vivo [e.g. 134, 347, 505] and the in vitro graft- vs-host reaction [509] (see review of previous documentations on the role in all these reactions [236]). The (relative) resistance of the immune system in thymectomized puberal mice implies three exceptions: 1. The nude mouse, congenitally deprived of the thymus [549]. This mouse does not reject allografts; its spleen cells are unable to induce 118 J. Comsa et al.

the graft-vs-host reaction and its hemolysin production after sheep erythrocyte injection is conspicuously low. Normal reactions are reestablished in all these tests by a thymus graft [398]. This topic will be discussed below (see Sect. 1.9.1). 2. The dwarf mouse [157,415]. 3.The thymectomized, lethally irradiated mouse, restored with bone- marrow cell transfusion [e.g. 367]. These animals survive both thymectomy and irradiation, but they do not produce antibodies against a large number of antigens, e.g., sheep erythrocytes [127,367] and skin allograft rejection is delayed [e.g. 361, 515]. This observation developed into a valid and generally accepted experimental design. It may still raise a problem when used to demonstrate the influence of thymic extracts. Indeed, the efficiency of a thymic extract was shown to tend towards zero in irradiated guinea pigs[ 114].This was not demonstrated by immunological testing; nevertheless, it could explain the inefficiency of thymic hormones in irradiated mice [306].

1.2 Cell-to-Cell Interaction in the Immune Response

Irradiated thymectomized mice, injected with lymphocytes from bone marrow or thymus, produced no antibodies when injected with erythrocytes (as demonstrated with the plaque-forming cell test [367]. Antibody production was normal in similar mice injected with both thymic and bone-marrow cells [e.g. 102]. This could not be observed when the thymic cells were killed by ultrasound [ 102] or by a specific antiserum. Newborn mice (CBA or C 57bl) were thymectomized, X-irradiated, and injected with sheep erythrocytes. Their antibody production was poor, as demonstrated by the plaque-forming cell test. It could be reestablished by injection of thymic and bone-marrow lymphocytes, but not with thymic cells alone [363,365,364]. Newborn thymectomized CBA mice were injected with syngeneic bone marrow and C 57bl thymocytes together with sheep erythrocytes. Then their spleen was searched for plaque-forming cells. If the test plaques were treated with anti-CBA serum, the number of plaque-forming cells decreased to near zero. Anti-C 57b 1 serum did not have a similar effect [363]. Thus, the antibody-producing cells differentiate from the receiver, but the (allogenic) thymus was necessary. In intact X-irradiated mice restoration of the immune response was possible with bone-marrow cell transfusions (plaque-forming cell test); in thymectomized irradiated mice it was not (reviewed in [3651). CBA mice were thymectomized, X-irradiated, and injected with thymic and bone-marrow cells either from CBA or T6 T6 donors (T6 T6 thymus Hormonal Coordination of the Immune Response 119

+ CBA bone marrow or the opposite). Their spleen cells were injected into second recipients thymectomized, X-irradiated CBA, and previously immunized against T6 T6 mice. In addition, the second recipient was injected with sheep erythrocytes. Three combinations were possible: 1. The second recipient mouse received spleen cells from a donor injected with T6 T6 thymus and CBA bone marrow. There was no net increase in plaque-forming cells in its spleen. 2. The second recipient received spleen cells from a donor bearing CBA thymus and T6 T6 bone-marrow cells. In its spleen there was an explo- sive increase in the mitotic rate, but no increase in plaque-forming cells. 3. The second recipient received CBA thymus and CBA bone-marrow cells. Mitosis and plaque-forming cells increased significantly in number [131]. This interaction between at least two cells for immune response was confirmed by numerous in vitro assays [e.g. 43] (see below). The two (B and T) cells were incubated in two compartments of a double diffusion chamber with sheep erythrocytes added to one compartment only. Antibody production was limited to the B cell compartment, but when B cells were incubated with sheep erythrocytes only, no antibodies were produced [3]. Similar observations were made with Shigella antigens [ 1871. In fact, the cooperation between T and B cells was found to be insufficient [e.g. 13,363]. Macrophages are also necessary [e.g. 185,201,208, 317, 379, 436, see review 282]. Two different T cells seem necessary for this interaction: those killed by antilymphocyte serum (long-lived) and those disappearing shortly after thymectomy [185].

1.3 Role of the Thymus in Immunotolerance

Immunotolerance was first observed in rabbits injected with antigen at birth [246]. This can be induced with repeated small doses or with a single massive dose of antigen. The effect of small doses was enhanced by cortisol injections [ 100]. Thymectomy was followed by a prolongation of immunotolerance beyond the normal time limit [104, 406, 493]. Thus, tolerance seemed to show just the absence of immunocompetent T cells. Still, these cells may be suppressed by thymectomy or by X-irradiation. In tolerant animals, thymectomy prolonged the tolerance period, whereas irradiation shortened it [ 101 ]. Apparently, cells (killed by irradiation) are necessary to maintain the tolerance. Thymectomized, X-irradiated mice bearing 120 J. Comsa et al. thymus grafts from tolerant donors [264] or infused with thoracic duct cells [99] or spleen cells [243] from tolerant donors became tolerant. This was confirmed in rats [237]. When the (tolerant)spleen or thoracic duct cells were treated with anti-Thy 1 serum prior to the transfusion, this passive tolerance was not induced. Tolerance still does not seem to be specific to T cells. Thymectomized, X-irradiated mice injected with bone-marrow and thymic cells became tolerant when either one of the two cells came from tolerant donors [98]. Enhancement of antibody production or tolerance depends on the ratio of T cells to B cells. Up to a certain limit T cells induce antibody production by B cells; over this limit they suppress it and may induce tolerance [ 184]. Be that as it may, the importance of the thymus in induction of immunotolerance can be demonstrated.

1.4 Role of the Thymus in Cytotoxic and Graft Immune Reactions

The allograft rejection is a reaction of the cellular immunity. Passive transfer of immunity against allografts was possible by cell transfer but not by injections of serum from an immunized donor [e.g. 372, reviewed 350]. An allograft is rejected at any rate by a normal animal, but receptors of immune cells rejected it as if they had been immunized already by a first graft (i.e., in about half the time required for the rejection of the first graft). Passive transfer of graft immunity by serum was also possible in rabbits [552]. Humoral immunity is supposed to play a part in allograft rejection [see review 4781. Allografts included in diffusion chambers were not destroyed [e.g. 8]. Divergent observations are scarce: lymphocytes of an immunized mouse were included in millipore membranes and implanted into an unimmunized animal. Skin grafts of the strain against which the lymphocyte donor was immunized were rejected by the receptor as second grafts [302,385]. Drainage of the thoracic duct in rats (i.e., deprivation principally of small lymphocytes) resulted in an significant delay in graft rejection [332]. Thymectomy at birth was followed by a long delay in skin graft rejection in mice [176, 192, 361]. The role of the thymus is essential: X-irradiated, thymectomized CBA mice injected with syngeneic bone marrow and C3H thymic cells normally rejected C 57 B skin grafts but not C3H grafts. Mice injected with anti-Thy 1 serum did not reject skin allografts [428] (see review of previous documentation [542]). Allograft rejection demonstrated the function of cytotoxic cells. Lymph-node cells from rats bearing a skin allograft kill kidney cells from the skin donor strain in vitro [ 541 ]. In cytotoxic cells [326], cytostatic activity (demonstrated by the inhibition of thymidine uptake) was distinguished from cytolytic activity Hormonal Coordination of the Immune Response 121

(demonstrated by the release of 51 Cr from the labeled target cells into the medium). Cytotoxic activity is supported by T cells [514]. It persists in lymphocytes where B cells have been discarded [e.g. 234]. It can be acquired in vitro by cultivation of T cells (from thymus, lymph nodes or spleen) with target cells [see 327]. This differentiation implies DNA synthesis [388]. Cytotoxic T cells are of a peculiar type, characterized by specific antigenic markers (see Sect. 1.7). They conserve their cytotoxic potential for months in vitro in the absence of the specific antigen. They have no "helper" function [see review 136]. This differentiation implies DNA synthesis [e.g. 388]. T cells are sufficient to manifest cytotoxic activity [94]. This observation was not confirmed. Thymocytes (i.e., immature cells) added to the medium, enhanced the cytotoxic activity of (mature) previously sensitized spleen cells from CBA mice against BALB lymphocytes [520]. In fact, cytotoxic activity was observed in a multitude of cells: T cells, B cells, null lymphocytes, and macrophages [93]. Macrophages are required for cytotoxic activity [e.g. 521 ], but macrophages alone manifest only cytostatic and no cytolytic potency [e.g. 241,274, 275]. The cytotoxic activity of macrophages may be induced by immunized T cells (immunized T cells conferred a cytotoxic potency to unimmunized bone-marrow cells) [e.g. 240]. Peritoneal macrophages from immunized mice were cytotoxic by themselves (supposed to be influenced by T cells in vivo [324]). It could be shown [84, 323] that the development of cytotoxic activity results from the synergy of two types of T cells, differing only in their antigenic markers (see Sect. 1.8). Cytotoxic activity needs the presence of helper T cells in much the same way as does the differentiation of B cells into antibody producers.

1.5 Suppressor T Cells

Spleen cells stimulated with a mitogen (concanavalin A) added to the medium will reduce the number of plaque-forming cells from spleen cells incubated with sheep erythrocytes [160]. T cells added to the medium induce a decrease in thymus-independent antibodies by B cells [e.g. 205, 206]. Injection of antilymphocyte serum in mice together with a thymus- independent antigen results in an enhanced production of antibodies [e.g. 35]. Mice were rendered tolerant against pneumococcal lipopolysac- charide by a massive injection at birth. Spleen cells of these mice, when added to the medium, will reduce the antibody production of normal spleen cells incubated with the same antigen to zero [ 152]. In nude mice (which produce thymus-independent antibodies in large amounts), the 122 J. Comsa et al. injections of thymocytes from their haired littermates together with thymus-independent antigen were followed by a comparatively reduced antibody production [259]. In these experiments the suppression of antibody production appears to demonstrate the activity of a peculiar type of "suppressor" cells. Mouse thymocytes incubated with X-irradiated spleen lymphocytes will develop into suppressor cells and inhibit the 3 H-thymidine uptake in a mixed lymphocyte culture. This characteristic is suppressed by pretreatment with anti-Thy 1 serum, suppressor activity is supported by T cells [167] but not by these alone. Immunosuppressor function could be demonstrated in macrophages [565]. The development of thymic cells in either suppressor or helper cells depends on the amount of antigen injected. In the spleen cells of mice, injection of 106 sheep erythrocytes induced the development of helper function, whereas injection of 109 erythrocytes induced suppressor function. Does this difference show that the same cells possess both potentialities, or does it demonstrate the selective development of one of those cell types? T cells were incubated with macrophages sensitized to sheep erythrocytes. These cells developed a suppressive influence when added to normal spleen cells, and a helper influence when added to spleen cells deprived of T cells [572]. The normal immune response may thus be the algebraic sum of the helper and the suppressor functions [see e.g. 204, 268]. These functions are supported by different cells. They differ in their antigenic markers. Helper cells bear the Ly 1 marker, whereas suppressor cells are labeled Ly 2, Ly 3 [78, 160, 163]. Two cells are necessary for the development of cytotoxic activity. It is still open to discussion whether suppressor and cytotoxic activity are of different types or two aspects of the same potential. Cytotoxic and suppressor cells bear the same antigenic marker, but suppressor cells are killed by cyclophosphamide [437] and a suppressor activity against cytotoxic cells could be demonstrated in an allograft test [105]. A distinction could be claimed between suppressor and cytotoxic cells [559].

1.6 Mechanism of the T Cell-B-Cell Interaction

Ever since the notion of T and B cells has been defined, speculation has started on the mechanism of their interaction. As a result hypotheses began appearing in great numbers. Just by the wording, these hypotheses seemed to have provided advanced knowledge, or even better, an illusion of knowledge, but in reality were based on absolutely no facts whatso- ever. These hypotheses are obsolete. Hormonal Coordination of the Immune Response 123

Recent investigation has demonstrated that T cells influence the B cells by soluble substances delivered into the medium. Spleen lymphocytes cultivated with sheep erythrocytes develop few plaque-forming cells when previously treated with anti-Thy 1 serum. If T cells previously sen- sibilized to sheep erythrocytes are added, the development of plaque- forming cells was significantly increased. The same effect was obtained by adding the supernatant of T-cell cultures [455]. In the incubation medium IgG and IgM antibodies were found [455]. Spleen cells from neonatally thymectomized mice (which normally do not differentiate into plaque-forming cells when incubated with sheep erythrocytes) develop plaque-forming cells when incubated with thymic lymphocytes or with the supernatant of their culture [e.g. 146, 235, 526]. Spleen cells from irradiated mice do not develop plaque-forming cells in a culture with sheep erythrocytes. This development is restored by addition of thymic lymphocytes from sensitized donors or by the supernatant of their culture medium [517]. Supernatants of activated T cell cultures induced plaque-forming cell differentiation in spleen cells of nude mice [211]. Mice were grafted with nonisologous skin. The regional lymph nodes were included in millipore chambers and implanted into intact mice of the first graft-bearer strain. The recipients rejected similar skin grafts in the time necessary for a secondary rejection [386]. Thymocytes from mice immunized against sheep erythrocytes were incubated with B cells (spleen cells treated with anti-Thy 1 antiserum) in the two compartments of a diffusion chamber separated by a millipore membrane. The B cells developed numerous plaque-forming cells, even when they came from unimmunized mice or from nude mice [ 186] or when a dialyzing membrane separated the two compartments instead of a millipore membrane [ 186]. Supernatants of T-cell culture immunized against Toxoplasma induced the production of anti-Toxoplasrna factor in nonimmunized macrophages [465]. The list of work on this topic is by no means exhaustive. Ten demon- strations were repeated over and over again with reasonably predictable results. Thus, T cellsinfluence B cells by a compound they deliver into the medium. The bulk of T cells can be defined to some degree as a dis- seminated endocrine gland. Apparently this compound is not specific, however, its production is induced by an antigen. Supernatants from mixed lymphocyte cultures could induce the development of the immune response of B cells against sheep erythrocytes [11, 160, 197, 440, 532]. Divergent observations were reported, however. Mice were immunized against synthetic peptidic antigens. T cells from their spleen were incubated and the supernatant together with the bone marrow was injected into X-irradiated mice. In the spleen of the recipients plaque-forming cells were found to differentiate from the erythrocytes coated with the 124 J. Comsa et al. same antigens, but not from the normal sheep erythrocytes [384]. This mediator secreted by T cells is probably not unique. Some of these factors are peptides with a mol. wt. ranging around 40 000 [ 186]; glycolipidic compounds with a similar action were also isolated [566,569,584]. The intimate mechanism of their action is unknown, but there is a suggestive parallel. Bacterial lipopolysaccharides mitogenic for B cells enhanced the immune response of B cells against sheep erythrocytes, a thymus-dependent reaction [276]. In fact, several mediators of different chemical nature were described. Ribonucleic acids from lymph nodes of rabbits bearing skin allografts injected into a normal rabbit induced in this animal the ability to reject similar skin allografts as if it had been immunized itself by a first graft [342]. Ribonucleic acids from spleen cells of immunized donors injected into thymectomized mice induced a normal immune response (plaque- forming cell test) in these animals [283]. Allograft rejection accelerated in rabbits injected with ribonucleic acid from lymph nodes of rabbits bearing an allograft [269, 342, 70]. (Still, ribonucleic acids are not supposed to be secreted into the medium.) The "suppressor" incubation medium of mouse spleen cells from donors rendered tolerant by a massive sheep erythrocyte injection contained a ganglioside which showed a suppressor influence in purified form [ 172]. Soluble mediators probably play a role in the interactions between macrophages and lymphocytes. Macrophages were incubated with an antigen. The supernatant of this culture induced a specific helper cell differentiation when added to the incubation medium of T cells [ 171 ]. Cytotoxic action was found to be mediated by soluble products of lymphocytes ("lymphotoxines" [527]). Development of cytotoxic activity in killer cells was found either to be dependent on a soluble product from helper cells [474] or enhanced by the supernatant of a mixed lymphocyte culture ("killing assisting factor" [393]). Supernatant of a lymphocyte culture from mice tolerant to human globulin was found to render mice injected with it tolerant [95]. This (unidentified) compound is secreted by T cells, since pretreatment of the lymphocytes with anti- Thy 1 serum sppressed the reaction. In conclusion, the intricate cell-to- cell interactions, ultimately resulting from the immune response, appear to be mediated by compounds delivered by the different cells into the medium. The intimate mechanisms of these reactions are unknown. Further information on the role of cAMP will be commented on in Sect. 6.1.3. The relation of the cell-to-cell activations mentioned above with the different hormones influencing the immune response awaits further research. Hormonal Coordination of the Immune Response 125

1.7 Distinction of T and B Cells

For many years it was believed that lymp,hocytes leave the thymus, which led to the conclusion that some of the lymphocytes circulating in the blood or fixed in lymphatic tissues may be of thymic origin. This was demonstrated first by two methods: 1. The consequences of thymectomy on the lymphocyte pool. Thymec- tomy was followed by lymphopenia and by atrophy of lymph nodes and spleen follicles [reviewed in 116]. In lymph nodes and spleen, the zones affected by this atrophy could be delimited (thymus-dependent zones [see e.g. 399, 400]). These zones include about 70% of the lymph nodes and 20% of the spleen. Lymphocytes in the thoracic duct are, for the most part, of thymic origin. After labeling the thymus in situ with 3 H-thymidine, about 0.02%-0.12% of the lymphocytes in the lymph nodes showed the label [533]. Thus, from one test to another the results were very different. 2. The description of thymic lymphocytes of intracellular enzymes dif- fered from those of the lymph nodes' lymphocytes such as alkaline phosphatase [50]. By this test, 10% of the circulating lymphocytes were identified to be of thymic origin, but no such lymphocytes were found in the lymph nodes. This research was placed on solid ground when the antigenic markers specific for the thymic lymphocytes [e.g. 424, 426, 429, 456] were identified, detectable by specific purified antibodies [e.g. 422, 427]. They were different from those directed against the antigenic marker of B cells [424]. These observations made it possible to describe with validity other differences between T and B cells: The surface of T cells is smooth, that of B cells villous [456]. Drugs such as phytohemagglutinin [e.g. 169] or concanavalin A induce an increased proliferative activity in lymphocytes of thymic origin only. It is possible to conclude from the phytohemagglutinin test that all circulating lymphocytes are of thymic origin [e.g. 145]. T cells and B cells have shown a different mobility in free-flow electrophoresis [e.g. 442, 545,556]. In fact, this could be expected. In free-flow electrophoresis of normal mouse lymphocytes two populations could be separated, whereas those of thymectomized, irradiated mice, restored with bone-marrow cells, migrated in a unique bulk [441 ]. In irradiated mice, injected bone-marrow cells migrate in the thymus, the bone marrow, and the lymph nodes. Thymocytes will not migrate into the thymus [ 193]. More elaborate techniques permitted the identification of several subpopulations in T cells: 126 J. Comsa et al.

1. By the antigenic markers. Within the thymus the Thy 1 antigen is highly concentrated on the surface of cortical lymphocytes, whereas in the medulla and on the circulating lymphocytes the markers Ly 1, Ly 2, and Ly 3 are highly concentrated, and Thy 1 is low [e.g. 84, 481]. The former are killed by an injection of cortisol in milligram dose, whereas the latter survive [65, 297]. 2. The cortisol-resistant cells correspond to the mature, immunocompetent T cells, Cortisol was injected in milligram amounts into a first mouse. Its thymus cells were transfused into an X-irradiated mouse together with sheep erythrocytes. Plaque-forming cells in the spleen of the irradiated receptor were about 20 times more numerous than after the transfer of the same number of thymocytes from a normal mouse [106]. Cortisol is known to kill about 95% of the thymocytes [see 147]. The cortisol-resistant cells in the medulla are derivatives of the cortisol-sensitive cortical thymocytes. When applied on the surface of the thymus, 3 H-thymidine labeled first the cortical cells, characterized by high density of Th 1 marker on the surface, then later the lymphocytes in the mid-cortex, and lastly those in the medulla, which are known to be cortisol resistant [533].

1.8 Subpopulations of T Cells

Antigenic differences seem to determine the function of the thymic cells. When leaving the thymus, different cells will sttle in the spleen and in the lymph nodes [456]. Research into antigenic markers may identify various subpopulations but of different significance: 1. Different stages in the maturation of T cells 2. Different types of mature T cells 3. Four successive stages can be distinguished. 1. Different stages in the maturation of T cells: a) The primitive (prethymic) lymphocyte located in the bone marrow. Its surface is smooth (not villous [499]. (Both smooth and villous lymphocytes were found in the spleen of nude mice as well.) It bears a weak Thy 1 [499] antigenic marker which is not dependent on the thymus (it was found in the spleen of nude mice [328, 435]) and in the bone marrow of thymectomized rats [262]. It is assumed that these cells are predetermined to migrate into the thymus when leaving the bone marrow. This seems to result from the observation of Nakao and co-workers [387]. Bone-marrow lymphocytes were separated by sedimentation. Those bearing a weak Thy 1 antigenic marker were Hormonal Coordination of the Immune Response 127

thus separated from the so-called stem cells. Injected into irradiated mice these cells, and these only, repopulated the thymus. The stem cells did not. Yet fetal liver cells, i.e., B cells (refractory to anti-Thy 1 antiserum), injected into splenectomized irradiated mice, repopulated in the thymus, lost their antigenic B-cell markers and became sensitive to anti-Thy 1 serum [214]. b) The proper thymocyte, located within the thymus, bearing a high concentration of Thy 1 antigen [e.g. 85,482]. c) The postthymic immature lymphocyte, still bearing a high density of Thy 1 antigen [482] and a low density of Ly 1, Ly 2, and Ly 3 antigens. They lose the TL antigenic marker when leaving the thymus [297]. All these cells are immunoincompetent. They acquire full immunocompetence at the periphery [482] or in contact with thymic epithelial cells [524]. d) The immunocompetent mature T cells. According to Stutrnan [482], mostly immature immunoincompetent lymphocytes leave the thymus. The few mature T cells within the thymus were said to remain (in grafted thymuses they were still of the donor type one year after the operation [4821 ). (For a review see [580, 5811 ). 2. Differences between mature T cells, related to their function: a) Helper T cells bear the Ly 1 antigenic marker, cytotoxic and suppressor cells the Ly 2 and Ly 3 marker [84, 86]. b) Helper and cytotoxic cells can be separated by sedimentation on a velocity gradient [513]. Suppressor and cytotoxic cells sedimented together and, since they are both Ly a- Ly 2÷, it is open to discussion whether they are different from each other. The fractionation by free- flow electrophoresis seemed to separate rather the immature port thymic precursors (small, sensitive to cortisol) from the mature T cells (larger, resistant to cortisol [ 1541 ). (For review see [205] ). In short, the differentiation of the different lymphocytes involved in the immune response appear to be in some way predetermined and not haphazard (those that do happen to reach the thymus on their migration from the bone marrow and those that do not). Still, all these cells circu- late in the same blood, and the hormonal influences they are subjected to may have different consequences from one cell to another.

1.9 Role of the Thymus in Organization and Generation of Immune Response (H. Wekerle)

The thymic cortex is a reticular sponge of epithelium-derived cells, which form sparse intercellular contacts via desmosomes. The wide gaps between 128 J. Comsa et al. the reticular meshwork are densely filled with small to medium-sized lymphocytes, those, particularly close to the reticulum cells, which display frequent mitotic activity. A possibly specialized epithelial cell of the thymic cortex, which might have a central part in T-cell maturation by engulfing certain lymphoid components, will be described below. Principally, the thymic cortex appears to be the location where most of the lymphoid proliferation occurs. Nevertheless, it is not fully clear why this proliferation occurs and what becomes of the cellular progeny which will not leave the thymus. The thymic medulla is composed of more solid epithelium, which ultrastructurally is distinct from the cortical reticuloepithelial cells. It is still unclear which maturation processes occur in this region. These processes appear, however, to be relatively late in the T-cell developmental pathway, since here most of the T lymphocytes, already immunocompetent, appear to be concentrated. Treatment of mice with high doses of corticoids results in a fast involution of the thymuses, principally of the cortex. Whereas in the cortex practically all lymphoid cells are lost within hours, the fewer medullary thymocytes are mostly preserved. Immunological function tests reveal that the cortisone-resistant thymocytes are immunocompetent, being responsive in lectin-induced mito- genesis, in transplantation, and graft-vs-host reactions. Generally, a pathway of lymphocyte migration can be observed in postnatal thymuses. Bone-marrow-derived, still incompetent pre-T cells invade the organs across the capsules. Having undergone the developmental steps leading to clonal diversity and propagation, the immunocompetent T cells appear to selectively leave the thymus via the high endothelial postcapillary venules to peripheralize into the lymph nodes and the spleen.

1.9.1 Immunological Functions of T Lymphocytes

Removal of the thymus during embryonic or perinatal stages has grave effects on the morphology and the function of the immune system. Inde- pendently Miller and Good found that thymoprivic animals do not reject foreign tissue transplants. Moreover, removal of the thymus impairs the capacity of the immune system to mount humoral antibody responses of the IgG type and to establish immunological memory. Delayed-type hypersensitivity reactivity and cellular immune responses against viral or related diseases are equally impaired. The depriving effects of thymectomy may be strong enough to prevent orderly growth of developing infant animals. Generalized wasting disease may be the cause of chronic infections by various agents. The impaired immune function corresponds to similarly grave changes in the lymphoid organ architecture. DeSousa et al. investigated the Hormonal Coordination of the Immune Response 129 histology of lymphoid organs from thymectomized mice. They found that the reduction of the organ sizes results mainly from the loss of lymphoid population of distinct fixed areas. The paracortical regions of the lymph nodes and the pericapillary cuffs of the spleeen were depleted. They are truly thymus-dependent areas, since restoration of the thymoprivic animals with transferred thymocytes also restores these areas. In contrast, other areas of the lymphoid organs, such as the primary follicles and the rims of germinal centers, are not affected by thymectomy, and are thus thymus-independent.

1.9.2 T Lyrnphocyte Differentia tion Markers

Thymus-dependent T lymphocytes are morphologically identical with the thymus-independent, bone-marrow-derived B lymphocytes. Both subgroups can, however, be distinguished by several typical markers on their surface membranes. B lymphocytes bear classic immunoglobulin on their membranes. In contrast, T cells lack surface immunoglobulins. In addition, Ig B cells, in most cases bear on their membranes not only Ig, but also receptors for Fc parts of extraneous immunoglobulins, and for C components. Most, but not all, of the T lymphocytes lack these receptors. Positive T cell markers have been identified following allogeneic immuni- zation. The most prominent among them is the Thy-1 antigen, the original 0 specificity. This antigen occurs in two allelic variants, Thy-1.1 characteristic for the AKR/J murine strain, and Thy-1.2 for most of the other strains. Thy-1 antigens are present during T-cell differentiation within the thymus as well as later on peripheral immunocompetent T cells. Moreover, it appears that low concentrations of Thy-1 are present on the membranes of pre-T cells, which have not been processed by the thymus. Except on T lineages, Thy-1 is found in brain tissue, and on certain myo- genic cell stages. In contrast to Thy-1 antigen, which is present on most sublines and differentiation stages of T cells, other T-cell markers are typical for limited stages or isolated functional sublines. Thus, they are true differentiation markers. Prime examples for intrathymic developing cells is the TL antigen system, which is not found on normal peripheral T cells, but on some thymus-dependent leukemias. Differentiation markers of peripheral as well as thymic, functionally definable subsets include antigens of the Ly series. Characteristic combinations of Ly-1 and Ly-2.3 antigens, which are genetically unrelated, are found on various T-cell subsets, such as T helpers, T suppressors, T killer cells, and T cells involved in delayed-type hypersensitivity (vide infra). Finally, certain T-cell sublines carry on their surfaces components of the major histocompatibility gene complex (MHC) or of associated genes. These include the I4 subregion products of the H-2 (the murine MHC system), which are found 130 J. Comsa et al. only on suppressor T cells; antigens of the Qa series, which have been recognized only recently, seem to appear only at various mature stages, indluding certain helper populations. These alloantigenic markers concern only the murine immune system. Analogous antigens are being recognized, however, also in other mammalian species, including man. This holds particularly true for the MHC- associated antigens and Thy-1 analogs. It should be mentioned, however, that human T cells exhibit an unique marker, namely receptors for untreated sheep red blood cells (SRBC receptors), a property which has been highly instrumental in isolating peripheral human blood T and B lymphocytes.

1.9.3 Role of T Lymphocyte Sublines in the Organization of the Immune System

As already mentioned, B lymphocytes are the precursors of the effector cells in the humoral immune response, the plasma cells. The role of the T cells is by far more complex. They include not only precursors for thymus-dependent immune functions, such as killer cells in the graft- rejection response, or in the delayed-type hypersensitivity, but possibly more intriguing is that the T-cell sublines effect the regulation and dosage of immune responses, both on the cellular and on the humoral level. There are T-cell populations which help, or increase, the effector function of either B cells or effector T cell precursors. On the other hand, other T-cell subsets, the suppressor T cells, counteract immune activation either by suppressing the activity of the effector cells directly, or more indirectly, by decreasing the activity of the helper cells required. Obvi- ously, helper and antagonistic suppressor cells are vital components of regulatory systems necessary for fine dosage and self-limitation of a given immune response, as will be discussed later. Thus, the original concept of clonal selection theories, which imply that a given antigen selects a passively waiting, preexisting lymphocyte clone with complementary receptors, is not totally tenable. Rather, the immune system appears to be a most complicated cybernetic network with many cellular components necessary to elicit one given response and to cause its termination. Obviously, these cells have to communicate with each other. Cellular communications within the immune system can principally occur either via soluble mediators, or via direct cell contacts, supposedly involving complementary receptor/ligand interactions. Both types of interactions have been described in the case of T cells and their reaction partners. Perhaps the best characterized mediator factor is the one emitted by suppressor T cells. It has been shown by Tada and his colleagues that suppressor T cells can be activated by antigen in a particular Hormonal Coordination of the Immune Response 131 context to release factor(s) which can suppressively act upon effector lymphocytes. These factors are remarkable insofar as they possess regions complementary to the eliciting antigen. Thus they include genuine antigen receptors. Moreover, the structure of these antigen-binding regions in many respects resembles that of comparable antibodies. The suppressive factors do not express, however, constant protein regions of immunoglobulins. They rather express determinants of the MHC and, specifically, of the I-J subregion, which has already been mentioned as being typical of suppressor T cells. There are claims that helper cells release analogous factors also characterized by antigen-binding regions and determinants of (non I-J subregion) loci of the MHC. These helper factors, quite obviously, would not suppress immune activation but would rather help its initiation and continuation. In addition to the antigen-specific, T-cell products involved in the regulation of the immune response, numerous antigen-unspecific factors have been identified, which can modulate effector activity. Among them, there are the T-cell replacing factor described by Schimpl and Wecker, which is thought to act in relatively late differentiation phases of B cell activation. Other factors modulate in vitro mixed lymphocyte reactions and the in vitro generation of T killer cells, and others appear to promote growth of single T lymphocytes to clones. Direct intercellular interactions are not as accessible as humoral interactions in terms of experimental investigations. There are, however, some stages of the ongoing immune response which undoubtedly depend on intercellular contact and recognition. The first stage of any immune reaction is recognition of the immunogen by the responding, complementary lymphocyte. A plethora of experimental data indicate that in the case of T cells mere binding of antigen to the complementary receptor regions is necessary (and this is true for many, if not all, T-cell sublines), however, it is by no means sufficient for recognition leading to T-cell activation and differentiation. The studies of Zinkernagel [587, 588] and Doherty, of Shearer, and of Rosenthal and associates showed that T cells have to recognize "their" antigen in the context of determinants of the self MHC. Thus, a virus-specific effector T cell would only recognize "its" virus when it is presented on a somatic cell of the same organism, thus posess- ing the same MHC antigens as the T cell. The same is basically true for the recognition of soluble or particulate antigen. Any antigen must be presented by an autologous cell, and this is done exclusively by the macrophage or by its functional sublines. It is still unclear how the associative recognition of antigen plus self recognition is effected by the T cells. The following principles seem to be valid. T-cell recognition aimed at eliminat- ing a membrane-expressed (foreign) antigen, such as virus-infected cells, is associated with corecognition of the classical transplantation antigens 132 J. Comsa et al.

(H-2K/D determinants in the mouse, HLA-A/B/C in humans). T-cell recognition, which leads to regulation and dosage of immune responses, in contrast, involves recognition of Ia determinants, which are equally coded for in the MHC, but which are predominantly expressed on lymphoid cells and macrophages in the body. The requirement of Ia antigen identity is also marked in reaction stages subsequent to antigen recognition. They may be the most important examples of contact interactions in the immune system. Ia antigen compatibility is apparently required for various stages of interactions between helper T cells and B cells as well as between suppressor cells and their respective targets, i.e., helper T cells and B cells.

1.9.4 Generation of Antigen Diversity in the Thymus

One of the most intriguing problems in cellular and molecular immunology is how the structural diversity of the antigen-complementary lymphocyte surface receptors is generated. For decennia, the core question was whether receptor diversity is precoded firmly in the genetic information ("germ-line hypothesis") or whether it is rather the result of somatic mutation. Recent experimental evidence, stemming from the work of Tonegawa et al., appears to favor a compromise. It appears now that most of the structural information of receptor amino acid sequences is, indeed, contained within the transmittable genetic information. It is, however, probably arranged in relatively small sequence fragments, which are somatically recombined to give rise to the structural diversity underlying the clonal complementarity. It is not known how and where exactly the cellular events, basic to molecular diversification, are happening. In the case of T cells, it is very probable that generation of diversity occurs within the thymus. This may be particularly evident in generating MHC determinant corecognition by developing T cells. Jerne has postulated that incompetent lymphoid precursors, which express receptors for self antigens, enter the thymus and start to interact there with their complementary "self" MHC determinants present in thymic stromal components. As a consequence, the primitive cells start to divide. In the course of proliferation, selective mutations were thought to alter the self-recognition, T-cell receptors to receptor structures complementary to some foreign structure. Indeed, Zinkernagel et al. reported of experiments which support part of this hypothesis. Their experiments suggest that the capacity of T cells to co- recognize self MHC determinants along with the foreign antigen structures is acquired while in the thymus, probably following a selective interaction with the thymic stroma. Wekerle and his colleagues very recently have found a new thymic reticular cell type, which could be involved in Hormonal Coordination of the Immune Response 133 that particular stage. These cells, thymic nurse cells (TNCs), are of epithelial origin as indicated by their ultrastructure and have the capacity to be invaded by viable T lymphocytes [585, 586]. Invasion appears to be the consequence of (self) recognition. Subsequently, the incorporated cells start to divide within their nurse cell. When the engulfed lymphocytes are completely surrounded by internal epithelial membranes, which have several specializations indicative of secretory activity, it is possible that the particular microenvironment may lead to guided differentiation. This could result either in receptor diversification or in ramifications of the specialized, functional T-cell subsets. Thymical hormonal factors could act particularly stringently in this environment. (In addition to receptor diversification, specialization into various functionally specialized T-lymphocyte subsets is assumed to occur within the thymus. It should be noted that incompetent precursor cells enter the organ and that their immunocompetent progeny leave it finally as specialized subsets, as indicated by functional tests as well as by suface marker analyses.)

1.10 Substitutive Therapy for the Thymoprivic Condition

Restoration of normal reactions in thymectomized animals (and in nude mice) was attempted by several methods and demonstrated by several tests. These tests are not equally significant. The most convincing is the survival test in animals known to waste and die following thymectomy, such as mice, hamsters, and guinea pigs. Certainly, this is only a basic test, and its results must be rechecked with specific immunological tests. However, thymic preparations previously tested on the survival of thymectomized animals acquire thus an outstanding validity when compared to those not tested in this way. This distinction is always made below.

1.10.1 Thyrnic Grafts

Wasting was prevented in thymectomized mice by thymic grafts [ 134] and their immune functions were restored [134, 486]. These mice rejected skin allografts normally [314, 318, 361 ], and their reactivity towards sheep erythrocytes (plaque-forming cell test)was restored [7, 130, 314]. This experiment was repeated over and over again with similar results. Conversely, immunocompetence of spleen cells in thymectomized rats (0 previously) was restored following their perfusion through a thymus [80]. The accelerated development of benzopyrene-induced skin tumor in thymectomized mice was abolished by thymic grafts [358]. Thymic grafts were able to restore the immunocompetence of thymectomized animals when enclosed in diffusion chambers [7, 212, 448, 487, 549]. 134 J. Comsa et al.

Incubation of mouse thymocytes with thymic epithelium resutted in an enhancement of their activity in a mixed lymphocyte culture [379]. This was tested by numerous methods [7,320, 346]. Grafts of thymuses deprived of thymocytes (by previous X-irradiation or by previous incubation in vitro [e.g. 252, 521, 524] were similarly active. In nude mice, thymic grafts, enclosed in diffusion chambers, induced a decrease in the lymphocytes bearing a (weak) Thy-1 marker [435]. These experiments support the supposition of a hormonal influence of the thymus.

1.10.2 Transfusion of Thymic or T Cells Wasting of thymectomized mice was prevented by transfer of thymocytes [e.g. 165,553]. These animals recovered normal immune potency. Similar results were obtained with transfer of spleen cells [e.g. 363,486]. In prepuberal thymectomized and X-irradiated mice this technique was extensively used to restore immune functions [e.g. 363, 127]. The increased sensitivity to polyoma virus in thymectomized mice was suppressed by transfer of spleen cells [3131. Every study has reported that the results of this therapy are transitory, although the influence of thymic epithelial grafts lasts as long as the graft itself. If the effect of thymocytic transfer results from the influence of thymic hormones, this could indicate that thymocytes carry the hormone but do not produce it themselves.

1.10. 3 Cell-Free Thymic Preparations The ideal way to attempt substitutive therapy in thymectomized animals was to use the supernatant of thymic epithelial cell cultures. However, to obtain this compound in amounts sufficient to prevent wasting in dozens of thymectomized mice is wishful thinking. This technique was limited to miniaturized tests under cell-culture conditions. A single injection of thymic culture supernatant accelerated the development of lymphatic organs in newborn mice [351 ]. The (weakly) Thy-1 spleen cells of nude mice acquired an increased reactivity in a mixed lymphocyte culture, when previously incubated on a monolayer culture of thymic epithelium [445]. The supernatant of thymic epithelial cultures, added to the incubation medium of spleen cells from nude mice gave them the capacity to differentiate into plaque- forming cells [307]. Mice were immunized against rat fibroblasts, thymectomized, and X-irradiated. Spleen cells from these mice, when injected into the hind paw of rats, induced only a very weak swelling of the popli- teal lymph nodes of the recipient. The swelling of the lymph nodes was significantly enhanced when the spleen cells were previously incubated Hormonal Coordination of the Immune Response 135 with the supernatant of a monolayer culture of thymic epithelium. After incubation with the supernatant of thymus culture, spleen cells from nude or thymectomized mice induced a graft-vs-host reaction and a sensitivity to concanavaline A. This was not the case with spleen cells from intact normal mice [3 24, 356]. Thus, the supernatant of a thymus epithelial culture proved to exert a specific influence on lymphocytes. It acted inasmuch as it substituted for the thymus.

1.10.4 Thyrnic Extracts

The simplest cell-free preparation is the supernatant of homogenized thymic tissue washed by an appropriate solvent (water, saline etc.) and separated by centrifugation. The first preparations were tested for their stimulatory influence on lymphatic cell proliferation in newborn animals [242, 351] and in rats made leucopenic by irradiation [83]. Injected into intact mice, the supernatant of a thymus homogenate induced an increase in labeled thymidine and glycocoll uptake of the lymphocytes in the thymus, the lymph nodes, and the spleen. Immunological tests with similar compounds are few. Supernatants of homogenized Sprague-Dawley rat thymus, injected into neonatally thymectomized Sprague-Dawleys bearing Wistar rat skin grafts, accelerated the rejection from 60 days (in untrated rats) to 12 days [81 ]. A crude aqueous extract from acetone- defatted calf thymus enhanced the antibody productions of irradiated rabbits [357]. A crude extract of rabbit thymus prolonged the survival and delayed the tumor development in thymectomized mice grafted with Lewis sarcoma [4631. Experimental testing of crude organ preparations is rarely satisfactory. The action of these preparations is always the expression of a sum of the actions of several compounds. This sum may be algebraic: protein fractions isolated from the thymus were shown to inhibit lymphocytic proliferation [289] and various aspects of the immune response [87,381, 405]. A dialyzable, nonpeptidic (pronase-resistant) immunosuppressive fraction could be isolated from calf thymus by precipitations with mer- cury nitrate [560]. The description of several extraction and fractionation methods made the use of crude thymic extracts very soon obsolete.

1.10.4.1 Purified Extracts Commonsense tells us that several truths must be emphasized from a biological point of view before discussing this topic. 1. An organ extract expected to contain a hormone is worth as much as the bioassay tests proposed for its activity. This truism is valid as long as the hormone is not obtained in a pure state and its composition is not known exactly. 136 J. Comsa et al.

2. One test is no test. Several tests may be valid and yet in contradiction to each other. They may have different meanings. 3. There is only one way to distinguish a hormone from a specific cellular constituent. By definition a hormone is secreted. This can be demonstrated by two experiments: a) The hormone (or its characteristic activity) is present in normal animals in the corresponding gland and elsewhere. b) Following extirpation of the gland, the hormone (or its activity) disappears entirely. There are two sorts of biological tests: a) Global tests. The typical example is the survival test, if available (as for the thymus in mice, hamsters, and guinea pigs). This, however, is only a first step, just demonstrating indubitably the existence of the hormone. b) More elaborate tests which elucidate the mechanism of the hormone action considered. These tests attempt to define which functions are lost following, e.g., thymectomy, and which thymic extracts are able to restore these functions. These tests make much use of isolated organs or cells. It is obvious that these tests performed in vitro are more convincing if the cells used come from thymectomized animals.

1.10.4.2 Peptidic Extracts Extracts Known to Ensure Survival of Thymectomized Animals: Thymosin (A.L. GoMstein and co-workers) Thymosin was obtained from a saline thymic extract by heating it to 80°C (precipitate discarded) and by successive precipitation with acetone and ammonium sulfate. Further purification was attained by successive chromatography on Sephadex, carboxymethyl cellulose, and diethylaminoethyl cellulose [222, 224, 258, 217,218]. A homogeneous peptide was thus obtained and its amino acid sequence determined [571]. Several consecutive reviews of this work were published [217, 218]. Of the thym- extomized mice injected neonatally with thymosin, 70% survived with no obvious disorders, whereas only 34% of the untreated mice survived. The dose injected was thus just below the optimal dose (which perhaps would restore the mice 100%). In these mice the blood lymphocyte count (decreased following thymectomy) was restored to (about) normal. Thymosin seems to exert a stimulatory growth influence on lymphocytes. It hastens the regeneration of lymphatic tissue following X-irradiation [220] or cortisol injections (in enormous amounts [450]). Thymosin was injected into neonatally thymectomized C57 B 1 mice at 1 mg daily (a partially purified compound). Spleen cells from these mice were injected into newborn BALB mice in which they induced graft-vs-host Hormonal Coordination of the Immune Response 137 reaction, whereas spleen cells from untreated thymectomized mice did not. Similar doses of spleen extracts injected were found to be inactive [315]. Since the spleen contains ten times less hormone than the thymus [ 121,329], this dose was probably insufficient. Injected into thymectomized mice thymosin enhanced the proliferation of their spleen cells in mixed lymphocyte culture [423]. Thymectomized mice injected neonatally with thymosin normally rejected skin allografts [219,243]. They rejected normally, after a transitory growth, sarcomas induced by Moloney virus [ 558 ], whereas there was no rejection of those tumors in neonatally thymectomized untreated mice. Conversely, injections of an antithymosin antiserum induced a significant delay in allograft rejection [248] .In thymectomized mice injected with antithymocyte serum thymosin had no effect on tumor aUograft rejection [4231. The influence of thymosin on humoral immune response in thymectomized mice was less significant [219, 248, 483]. (Nevertheless, thymosin injections could not restore the ability of neonatally thymectomized mice to reject allografts or their spleen cells to induce a graft-vs-host reaction in newborn mice [4831.) In thymectomized, X-irradiated rats (900 rad) reconstituted with isologous bone-marrow thymosin injections had no influence on lymphatic tissue regeneration and antibody production [306]. We should recall that in irradiated guinea pigs the influence of thymic hormone tended toward zero [ 111 ]. In nude mice, thymosin injections induced an increase in the number of plaque-forming cells after an injection of sheep erythrocytes [63]. When incubated with thymosin, spleen cells of nude mice developed plaque-forming cells [ 11 ]. Incubation of bone-marrow lymphocytes with thymosin resulted in the appearance of antigenic markers specific for T cells on the spontaneous rosette-forming cells [30]. The enzyme terminal deoxynucleotidyl-transferase, characteristic for thymocytes, was induced in bown-marrow cells of thymectomized or nude mice [401]. In humans suffering from immune deficiency thymosin was largely used. The first case report (as far as we know) was that of a girl suffering from almost constant recurrence of infections constained for years with antibiotics. She was unable to develop positive cutaneous reactions after an infection (Candida sepsis) or vaccination (mumps) or to produce antibodies when challenged with various antigens. Spontaneous rosette-forming cells in her blood were significantly rare. All these abnormalities were suppressed by repeated thymosin injections [529]. Thymosin therapy for immunodeficient patients resulted in an improved general condition, an increased blood lymphocyte count, and in an increased number of rosette- forming cells [225,529]. In cancer patients similar results were obtained on lymphocyte count and the number of rosette-forming cells. 138 J. Comsa et al.

"The clinical response must be considered anecdotical" [217, 447]. Incubation with thymosin induced the development of antigenic markers in human lymphocytes (from healthy subjects and one cancer patient [503]). Incubation with thymosin of circulating human lymphocytes resulted in an increase in the number of rosette-forming cells [528]. The same influence was found with fetal calf serum [82]. The authors consider this observation as (primary) evidence for the delivery of thymosin into the circulation. Still, serum of nude mice has shown the same influence (T.D. Luckey, unpublished communication). Thymosin Activity Tested In Vitro. Thymosin was added to the incubation medium of lymphocytes. The cells, thus treated, were tested for their degree of differentiation and maturation. Incubation with thymosin induced the appearance of antigenic markers of thymocytes in mouse bone-marrow lymphocytes, in lymphocytes from nude mice or from 14-day-old fetuses [295]. The maturation of spleen cells from mouse fetuses and newborn mice was hastened (as demonstrated by their ability to induce graft-vs-host reaction and their behavior in mixed lymphocyte culture) by previous incubation with thymosin [ 108, 221 ]. Bone-marrow cells from newborn mice were incubated with thymosin and injected into thymectomized, X-irradiated mice together with normal bone-marrow cells. In the spleen of the recipients, normal numbers of plaque-forming cells were found, just as if they had received bone marrow and thymocytes [359]. In bone marrow incubated with thymosin, the number of cells killed by an (brain!) anti-Thy 1 antiserum increased [358, 503]. Lymphocytes from nude mice, from fetal mouse fiver or from bone marrow differentiated into T cells as evidenced by specific antigenic markers, by their ability to ensure helper function and to form T-cell rosettes with sheep erythrocytes [451]. Thus, thymosine was proved to be active in thymectomized and in congenitally thymoprivic mice. This is demonstrated by the survival test and by a large range of immunological tests. Extracts from a culture of thymic epithelial cell, prepared with the thymosin method, showed immunostimulant properties similar t-o those of thymosin [563]. But it must be said that the survival test was performed only once with a very crude preparation. The last steps of purification were only tested by the rosette test, and the reliability of these results may be questionable (Luckey and co-workers). Thymic Hurnoral Factor ( Trainin and co-worker) Preparation [301,508, 506]. A centrifuged, thymic homogenate in 0.1 M phosphate buffer at pH 7.4 was fractionated by successive dialyses (dis- carding by the first molecules of 120 000 and by the second molecules of 6000). The second dialysate contained the hormone in purified form. Precipitation by ammonium sulfate was not carried out. Hormonal Coordination of the Immune Response 139

Injections of the thymic humoral factor reduced the incidence of wasting in neonatally thymectomized mice to 20% (from 60% in untreated mice [ 510] ). This treatment prevented thymoprivic atrophy of lymphatic tissue and the decrease in the number of circulating lymphocytes. Thym- ectomized mice thus treated rejected skin allografts normally [507, 505]. The authors made much use of the in vitro graft-vs-host reaction test [22]. Spleen cells from neonatally thymectomized mice were found unable to induce the growth of an allogenic spleen fragment when added to the medium. This capacity was restored by repeated injections of thymic humoral factor [505]. Spleen cells from thymectomized mice acquired the graft-vs-host ability when incubated with thymic humoral factor [509]; such treatment also increased the survival rate of thymocytes when cortisol was added to the medium. Thymocytes incubated with thymic humoral factor induced the in vitro graft-vs-host reaction. In other words, they behaved like the few mature T cells present in the thymus, which are also resistant against cortisol [511]. This indicates that thymic humoral factor is involved in some way in the maturation process of thymocytes. This supposition was substantiated by further experiments. Prepuberal mice were thymectomized, X-irradiated, restored with bone marrow, and injected with sheep erythrocytes. Spleen cells from these mice did not differentiate into plaque-forming cells, even when the mice received thymic humoral factor injections. The normal reaction was restored when the mice received injections of thymocytes together with the hormone, or thymocytes preincubated with the hormone [439]. Mice were injected with anti-lymphocyte serum. Spleen cells from these mice did not induce the in vitro graft-vs-host reaction, even when thymocytes were added. The reaction was restored when thymocytes and thymic humoral factor were added together [327]. Lymphocytes from thymectomized mice reacted poorly in mixed lymphocyte cultures. Normal reaction was restored when thymic humoral factor was added. The addition of thymic humoral factor had no effect on lymphocytes from thymectomized, X-irradiated mice, unless thymocytes (inactive by themselves) were added together with the hormone. Thymic cells from a mouse, previously injected with 5 mg of cortisol, enhanced the mixed lymphocyte reaction by themselves, but in this system thymic humoral factor had no additional influence [516]. This indicated:

1. That the target of thymic humoral factor as the immature thymocytes, in which it induces the maturation to T cells. 2. That the cells able to respond to thymic humoral factor are in some way predisposed to do so. (Could this explain the inactivity of the thymic hormone in irradiated animals mentioned above?) 140 J. Comsa et al.

Mouse fetal liver lymphocytes, i.e., B cell precursors (only?), incubated with thymic humoral factor, induced the in vitro graft-vs-host reaction [2"13]. Yet, spleen cells from thymectomized mice preincubated with thymic humoral factor did not induce the in vitro graft-vs-host reaction [486], and thymic humoral factor did not induce transplantation immunity in nude mice [408; see also 579]. The mechanism for the action of thymic humoral factor is not known.

Homeostatic Thymic Hormone (Comsa and co-workers) Preparation [see 113], from a crude acidic extract (H~ SO4 M) fractionated successively by ammonium sulfate precipitation and isoelectric precipitation in 60% alcohol solution (pH 7 for impurities, pH 6.2 for the active fraction) a homogeneous compound was obtained by successive chromatography on Sephadex and hydroxyapatite (elution with potassium phosphate buffer at pH 6.75 in a molarity gradient). Wasting in thymectomized guinea pigs was prevented by using partially purified thymic extract and a purified fraction of this same extract [see 113]. It was a complete success, since all thymectomized guinea pigs thus treated survived in good condition, whereas only 60%-70% of neonatally thymectomized mice were preserved from wasting by administering thymosin or thymic humoral factor injections. Wasting after thymectomy is less frequent in guinea pigs (50%-60%) than in mice (70%, according to A. Goldstein and co-workers). In rats thymectomized at 30 days, injections of homeostatic thymic hormone prevented the consequences of thymectomy on the endocrine glands and on lymphatic tissue [see 1131. Circumstantial evidence was obtained to support the supposition that this hormone is secreted. It was found in the thymus, in the lymph nodes, and in the spleen or normal prepuberal rats, in the ratio 10:3:1 [124]. Three days after thymectomy, it disappeared from the lymph nodes and the spleen [121]. Its secretion is influenced by the adenohypophysis, the adrenal cortex, the thyroid, and the gonads (see Sect. 6.2). Immunological tests performed with this compound are scarce. In infantile thymectomized guinea pigs, it restored the antibody production against HO Salmonella antigen [ 115]. In thymectomized and hypophysectomized rats it restored the influence of hypophyseal growth hormone on antibody production [125] and on allograft rejection [123]. The acceleration of allograft rejection, induced by corticotropin, was suppressed by a simultaneous injection of thymic hormone ("The thymic hormone is immunostimulant and immunosuppressive. It all depends on the hypophyseal hormone it has to face") [ 123]. Thymosin or homeostatic thymic hormone delayed the death of mice injected with a supernatant of Yersinia pestis culture [141]. This may indicate an immunostimulant influence, among others. Hormonal Coordination of the Immune Response 141

With the allograft rejection test it could be seen that the thymic hormone is only needed for the primary reaction: Skin allografts were rejected by normal rats within 7.1 -+ 1.0 days and by thymectomized rats in 9.8 -+ 1.3 days. Second grafts implanted in those rats were rejected in 4.6 --- 0.5 and 4.4 --- 0.4 days, respectively. The hormone of Bernardi and Comsa exerted a chemotactic influence on lymphocytes. However, it cannot be said on which one it was [ 125]. This has been presumed [349, 245, see review] with no direct evidence. Considering the intricate interactions between the homeostatic thymic hormone and other hormones, this topic will be resumed below (see Sects. 2-6).

Thymic Polypeptidic Hormones Not Tested with the Survival Tests Thymopoietin ( G. GoMstein and co-workers) Preparation. An extract with 0.1 M ammonium carbonate was fractionated by heating to 70°C, dialyzed, and successive chromatographies on Sephadex and hydroxyapatite and polyacrylamide electrophoresis were carried out [228]. By this method two different homogeneous fractions were obtained, both active in GoMstein's test. Both peptides were obtained in a pure state and their amino acid sequence was determined [461]. The starting point of this work was the observation of an accelerated neuromuscular transmission in thymectomized animals, which can be prevented by thymic grafts [230]. Conversely, the neuromuscular chronaxy was found to be decreased in thymectomized rats [420]. From calf thymus, an extract could be prepared which, by repeated injections, induced a neuromuscular block in guinea pigs [226]. The neuromuscular disorder induced by this compound (named thymin, now called thymopoietin) is described in detail in [231]. The guinea pigs served as a basis for bioassay using thymopoietin. Culture of bone-marrow lymphocytes or of the B-cell fraction from mouse spleen lymphocytes with thymopoietin added resulted in the appearance of TL and Thy 1 antigenic markers [42,296]. Spleen lymphocytes from nude mice treated with thymopoietin acquired the antigenic markers TL and Thy 1 and developed a helper influence toward B cells, as shown by the plaque-forming test [450]. In the B-cell fraction of mouse spleen lymphocytes, the addition of thymopoietin to the medium induced the appearance of TL antigenic marker [43]. The cells containing terminal deoxynucleotidyl-transferase from mouse bone marrow are lysed by anti-Thy 1 antiserum after pretreatment with thymopoietin (in other words, they differentiated into prothymocytes [232]). In a one- way mixed mouse lymphocyte culture, thymopoietin induced an increase in the cyclic AMP content [488]. In thymocyte precursors thymopoietin, 142 J. Comsa et al. added to the medium, induced the appearance of antigenic markers specific for T cells (TL, Ly 1, Ly 2, Ly 3, Ly 5). But thymopoietin was unable to restore the normal reaction to PHA 0.2 concanavalin A of lymphocytes from thymectomized mice [574].

Serum Thymic Factor (Bach and co-workers) The compound was isolated from blood serum by two dialyses (active fraction is dialyzed in the first step and retained in the second) the successive chromatographies on Sephadex and carboxymethyl cellulose, thin-layer chromatography, and high-voltage electrophoresis were carried out [32]. The composition of the serum thymic factor was determined [ 129, 417]. (For an extensive review see [27] ). Bioassay of this compound is performed by the azathioprine test. Aza- thioprine, added to the medium, killed few of the spontaneous rosette- forming cells from mouse bone marrow but killed significantly more rosette-forming cells from bone-marrow cells preincubated with thymosin [30]. This test was further developed [33]. According to the following descriptions, azathioprine was added to the medium: At 1 ug/ml it totally suppresses rosette-forming cells from the thymus and up to 20% from the spleen. At 50/~g/ml it suppresses 70% from the lymph nodes. At 100 ug/ml it suppresses up to 20% only from the spleen of thymectomized mice. Mice were thymectomized or injected with anti-Thy 1 antiserum. The rosette-forming cells did not decrease in number. However, the azathioprine-sensitive cells decreased and the azathioprine-resistant cells increased. Incubation of these cells with thymosin or normal mouse serum resulted in an increase in the number of azathioprine-sensitive cells. Serum from thymectomized or from nude mice did not have this effect [26]. Thus, the highly azathioprine-sensitive, rosette-forming cells were presumed to be of thymic origin. Spontaneous rosette-forming cells from bone marrow are insensitive to anti-Thy 1 antiserum. Preincubation with normal mouse serum resulted in the appearance of spontaneous rosette- forming cells sensitive to anti-Thy 1 antiserum in bone-marrow cells from normal, thymeectomized, or nude mice. Nude mouse serum did not have this influence. This potency of normal mouse serum decreased by half within 150 min following thymectomy, remained constant at this decreased level for 1 week, and began slowly decreasing again later on [28]. The serum thymic factor was found in human serum. Its level is high in children and decreases with age. It decreased to zero in a patient thymectomized for myasthenia gravis [33]. Hormonal Coordination of the Immune Response 143

The serum thymic factor is prepared from pig serum (the serum thymic factor content decreased in the serum following thymectomy in pigs [309]. Serum thymic factor, added to the medium, increased the resistance of thymocytes against cortisol (circumstantial evidence of their maturation). It induced the appearance of specific T-cell antigenic markers and an increase in cyclic AMP content in these cells [21 ]. In normal mice, one single injection of serum thymic factor increased about 5-8 times the number of rosette-forming spleen cells [561]. The validity of induction of antigenic markers, characteristic ofT cells, as a test for thymic hormone action cells was questioned. Thus differentiated, they did not necessarily show T-cell properties in other tests, such as helper function or graft-vs-host reaction. Only thymopoietin, thymosine, and thymic humoral factor were found to be able to induce in vitro a total T-cell maturation [479]. Furthermore, serum thymic factor injections in thymectomized mice did not restore the normal reaction of their lymphocytes to phytohemagglutinin and to concanavalin A [574]. A demonstration of thymic origin of the serum thymic factor was attempted [33]. Grafts of thymic epithelium (thymomas or thymic grafts borne 8 days previously by a first recipient, in which thymocytes are known to be dead), enclosed in millipore chambers, were implanted into neonatally thymectomized mice. In the spleen of these mice the azathioprine-sensitive, rosette-forming cells increased in number. This proves that thymic epithelium exerts its influence through a millipore membrane. Nevertheless, some doubts arose as to whether serum thymic factors are secreted by the thymus. A thymic extract was prepared from calf thymus using Bach's method. This extract did not induce the development ofT-cell rosettes when injected into nude mice. On the other hand, serum thymic factor activity was found in similar calf thymus extracts when this factor was added to the thymus homogenate before the extraction. Thus, the extraction did not destroy the serum thymic factor presumably contained in the thymus [2841. This observation rejects by no means the idea that the thymus is involved in the production of serum thymic factor. Its site of production remains, however, to be found.

Lymphocyte Stimulating Hormone (Luckey and co-workers) A saline thymic extract was precipitated with 20% ammonium sulfate. The precipitate was dissolved in water and precipitated with methanol. The redissolved precipitate was fractionated by successive chromatography on diethylaminoethyl cellulose and Sephadex. It was proved homogeneous by polyacrylamide electrophoresis [329,434]. The original bioassay test for monitoring the extraction and purification is based essentially on the acceleration of the development of the lympho nodes in newborn mice [351]. The influence of this compound 144 J. Comsa et al. on the immune response was demonstrated. Injected into new-born mice, the fraction known to induce the accelerated lymph node maturation induced also the increase in the number of plaque-forming cells against sheep erythrocytes in the spleen of unimmunized newborn mice (back- ground plaque-forming cells) and of newborn mice injected with sheep erythrocytes [91, 247, 434]. From the impurities discarded by electrophoresis, a fraction was identified which antagonized the lymphocyte- stimulating factor. Lymphocyte-stimulating activity was found in calf spleen extracts prepared and fractionated by Luckey's method (about six times less than that from calf thymus). This may be correlated with the observations of Comsa and co-workers [ 124, 121] who found a roughly similar relation in the rat thymus and spleen content of homoeostatic thymic hormone.

Thymus Polypeptide Fraction (Milcu and Potop) The preparation is described in [334]. It is a crude extract, and although not tested with the survival test, it was proved active in thymectomized rats with an impressive number of metabolic tests [356]. Its influence on the immune response was demonstrated by the observation of an increased antibody production in hamsters injected with influenza virus and in X-irradiated rabbits injected with HO Salmonella antigen. As far as we know, the influence on immune response has not been tested in thymectomized animals. This extract was shown to delay the development of malignant tumors induced by carcinogens (such as methylcholanthrene or dimethylamino- azobenzene) or grafts (Guerin's tumor) in rats. This was also observed in cultures from a cancer derived from buccal epitheliums (KB tumor) [356]. It was also alluded to above that there is a delayed influence of the thymus on cancer development and an acceleration of cancer development in thymectomized animals. This has generally been interpreted as a thymic influence on cancer immunity. The observations made on cancer cultures hardly fit into this hypothesis.

1.10.4.3 Lipidic Extracts The first attempt to prevent the consequences of thymectomy in guinea pigs with a lipidic thymic extract was mede by Bomskov and co-workers [see 113]. It was a total failure. Isakovic and co-workers [270] extracted calf thymus with chloroform and methanol. The extract, suspended in saline, was injected into neonatally thymectomized rats. This treatment prevented the delay in growth and the decrease in lymphocyte count following thymectomy. Rats, thus treated, were immunized against bovine serum albumin at 60 days, and the delayed hypersensitivity test was performed 18 days later. The reaction Hormonal Coordination of the Immune Response 145 decreased in untreated thymectomized rats and was normal in those injected with the extract.

The Thymosterines (Potop and Milcu) Calf thymus was extracted with acetone and the residue was extracted with ether. Phospholipids were discarded by cold acetone from the dried etheric extract. The residue was fractionated by successive chromatographies [see 421 for details]. First fractionation separated two fractions: III B stimulating and IIB inhibiting tumor development. By several chromatographies of IIB a steroidic fraction was obtained (fraction S) strongly inhibiting tumor development. This fraction was named Thymosterin. Fraction S inhibited in vitro the development of KB tumor and human thyroid cancer cultures. In vivo, it delayed the growth of carcinogen-induced and grafted tumors, such as Jensen's, Walker's, and Ehrlich's sarcoma and Gu+rin's adenocarcinoma. Tumor grafts in thymosterin-injected rats showed signs of regression, such as necrosis and fibrosis. Walker sarcoma grafts decreased in size in thymosterin-injected rats, and these rats survived without exception when all untreated tumor-graft rats were dead. The influence of this extract on immune response was tested on mice injected with sheep erythrocytes. Hemolysin titers and plaque-forming cell development were significantly increased in mice injected with the partially purified fraction IIB. Thus, the influence of thymosterin on tumor development in vivo can be understood as expressing the stimulation of immune functions. The influence of thymosterin on tumor development in vitro cannot. The immunostimulant action and the retardant action on tumor cultures are certainly two different phenomena. Since the immunostimulant effect was demonstrated with the partially purified fraction B (and, as far as we know, not with the presumably pure thymosterin), it is open to discussion whehter these two actions express the activity of the same chemical entity.

2 The Adrenal Cortex

Adrenalectomized and normal rabbits were injected with typhoid vaccine. The adrenalectomized animals produced three times more antibodies [267]. Normal and adrenalectomized rats were injected with sheep erythrocytes. Lymphocytes from their lymph nodes and spleen were incubated and the hemolysin released into the medium was determined. It was lower in adrenalectomized rats [433]. Thus from the very start the observation appeared controversial. 146 J. Comsa et al.

Adrenalectomy was said either to result in an accelerated rejection of skin allografts in mice [e.g. 90, 319, 353] or to show no influence [48, 351]. Still, these observations raise some doubt. The adrenalectomized mice were "protected" from the consequences of adrenalectomy on electrolyte metabolism by repeated injections of desoxycorticosterone in large amounts (1-2.5 mg once a week, as microcrystalline suspensions). This is a very important source of error. Strictly speaking, the authors never saw a truly adrenoprivic mouse. Allograft rejection was slightly accelerated in adrenalectomized mice protected just by NaC1 added to the drinking water [89, 90, 238]. In Sprague-Dawley rats grafted with DA rat skin (a H 1 incompatibil- ity) adrenalectomy resulted in a slight delay in skin allograft rejection from 7.1 -+ 1.3 days in normal rats to 12.2- 1.5 days in adrenalectomized animals. Additional thymectomy was followed by an additional delay (to 15.9 -+ 1.6 days). In Sprague-Dawley rats grafted with DB rat skin, adrenalectomy had no influence on the graft rejection [48]. Most of the experiments on the influence of the adrenal cortex on the immune response were performed on normal animals injected with large doses of corticosteroids (see [576] for a recent review). The earliest experiments, performed with adrenocortical extracts of an unknown composition, showed that repeated injections of these extracts, together with, before or immediately after the injection of an antigen into normal rabbits [96, 195, 18] enhanced antibody production. This was explained as a result of the well-known deleterious influence of adrenocortical hormones on lymphocytes [147]. The increased amounts of antibodies in the serum were supposed to be a result of their release from the lysed lymphocytes. This explanation is not exhaustive. The lysis of lymphocytes takes place within hours after corticoid injection, whereas the increase in the antibody level in the serum is gradual, occurring over several weeks [96]. In addition, the mature, differentiated lymphocytes, participating in the immune response, are not lysed by corticoids [e.g. 106, 107]. Cortisone or cortisol injections in milligram amounts, before or together with the antigen but not afterward [166], were shown to result in a decreased production of antibodies [e.g. 52, 53, 61, 143, 153, 179, 190,200,203,250, 253,335,341,352, 373]. In mice, injection of 2.5 mg cortisol resulted in a decrease of in vitro spontaneous killer activity of their spleen cells [257]. In mice, IgM and IgG hemolysin production is totally suppressed when cortisone is given 4 days previous to the antigen (sheep erythrocytes). Administered after the antigen, cortisone injection results in suppression of IgG antibodies only [ 168]. In previously immunized rabbits, a single cortisone injection suppressed the secondary response [ 191 ]. Sheep erythrocytes are lysed with a significant delay by macrophages of rabbits injected with cortisone Hormonal Coordination of the Immune Response 147

[280]. In frogs, cortisone injections were followed by an increased antibody production [60]. In all these experiments cortisone or cortisol was administred in large, arbitratily chosen, doses. A quantitative relation between the injected corticoid and the injected antigen dose could be roughly demonstrated: in rats, cortisone injections (4 mg) suppressed the anti-sheep-erythrocyte antibody production. This could be restored if the antigen dose was increased a 1000-fold [54]. In mice, the production of hemolytic antibodies against sheep erythrocytes was totally suppressed by an injection of 5 mg corticosterone, whereas 1.0 mg or 2.5 mg were followed by a partial suppresion only. Desoxycorticosterone was found to be inactive [389]. In rats, single injections of 5 mg desoxycorticosterone were followed by an enhanced antibody production [97]. Allograft rejection was delayed by cortisone injections in rabbits [58], guinea pigs [476], and rhesus monkeys [305]. Desoxycorticosterone was inactive [305]. Aldosterone injections also delayed the allograft rejection in rats but so did spironolactone [48]. Metopyrone inhibited the in vivo graft-vs-host reaction [1]. Thus, corticoids and corticoid antagonists showed similar effects. The development of grafted [5, 374] or virus- induced [3451 tumors was accelerated by cortisone injections. In all these experiments, steroids were administered in milligram amounts to intact animals. These observations were not entirely confirmed in doses closer to normal conditions. Adrenalectomized or adrenalectomized and thymectomized Sprague-Dawley rats were grafted with DA rat skin. They were implanted with pellets of aldosterone, corticosterone, desoxycorticosterone or cortisol, alone or in combination. They resorbed daily 1 /ag of aldosterone or 130/~g of the other steroids. Those are mounts just sufficient to ensure survival. In these animals: Adrenalectomy was followed by a slight delay in the allograft rejection (from 7.1 -+ 1.3 to 12.2 -+ 1.6 days). Additional thymectomy was followed by an additional delay (15.8-+ 1.3 days). Implants of aldosterone, corticosterone or desoxycorticosterone partially suppressed this delay (8.5-10.0 days) in both adfenalectomized and adreno-thymectomized animals. Implants of two steroids reestablished at least the normal rejection period (6.7-7.0 days). A third implant had no additional influence. Cortisol implants were by themselves inactive, but additional cortisol implants delayed the rejections in animals implanted with one or several of the other steroids [ 120]. There was no difference between the corticoid effect in adrenalectomized and adreno-thymectomized rats. Furthermore, in adreno-thym- 148 J. Comsa et al. ectomized rats injected daily with the Bernardi-Cornsa thymic hormone preparations, this additional treatment had no influence on the allograft rejection [ 117]. As soon as corticoids were administered, the influence of the thymus disappeared. From this experiment we can identify under physiological conditions: (a) three immunostimulant corticosteroids (aldosterone, corticosterone, and desoxy.corticosterone) and (b) a minor immunosuppressive steroid, cortisol (this is not exactly under physiological conditions, since rats do not produce cortisol). This conclusion is oversimplified. Under adequate conditions all corticosteroids known thus far have shown an immunostimulant effect. Rabbits were immunized against bovine serum albumin or diphtheria toxoid, and 40-100 days later their lymphocytes were incubated with sheep erythrocytes coated with the same antigens. These erythrocytes were lyzed if the medium contained at least 1 ng/ml and less than 0.1 gg/ ml cortisol [ 10]. Without added cortisol, the hemolysis occurred only if the incubation medium contained 25% fetal calf serum.In higher concentrations cortisol added to the medium suppressed antibody production in vitro [109], [e.g. 380], cortisone (70 #g/ml was ineffective on the in vitro antibody production of immunized lymphocytes [ 179]. It should be noted that metopyrone injections inhibited the graft-vs-host reaction in rats [ 1]. In conclusion, the dose-response curves of corticoids on the immune response appear to be bell shaped. Cortisol acts as an immunostimulant at 1 ng/ml of medium and as an immunosuppressive at 0.1 #g/lnl [ 10]. Aldosterone acts as an immunostimulant at 1/~g/day [ 117], as a partially immunosuppressive at 1 rag/day, and as a totally immunosuppressive at 5 mg/day [389]. Desoxycorticosterone still acts as an immunostimulant at 5 mg/day [97]. The immunosuppressive dose range is unknown. Perhaps it exists since desoxycorticosterone was prophlogistic at milligram doses and antiphlogistic at 25 mg/day [432]. This is, of course, a rough estimate. The results may differ within enormous limits according to the test used. For instance, cortisol was found to be immunostimulant on incubated human B cells when added in such enormous concentrations as 10 s [567]. Stress was generally followed by a depression of the immune response. This was obtained by overcrowding [473, 518], by training to avoid elec- troshock announced by a light-signal [473, 547], by adaptation to high altitues [77], by ether anesthesia [207] or by short exposure to a temperature of 37°C [416]. In fact, the observations are not easy to interpret. During the general adaptation syndrome, distinguishable stages should be considered: (a) the alarm stage, lasting several hours, and characterized Hormonal Coordination of the Immune Response 149 by a relative adrenocortical insufficiency; (b) the resistance stage, lasting for weeks, characterized by an enhanced corticoid production and by symptoms of hyperadrenocorticism and (c) the terminal exhaustion stage, characterized by adrenal deficiency. Thus, the effects of stress are certainly different in terms of duration. Examination of the adrenals is a necessary complement to those experiments. It was performed by Gisler [207] who found an approximately twofold increase in the plasma corticosterone level in mice following ether anesthesia. A detailed dose- response curve of corticosterone on immune response would be useful. The experiments quoted above simply indicate the possibility of plotting this curve. It is generally believed that corticosteroids inhibit the immune response inasmuch as they kill cells [e.g. 147, 150]. Which cells are killed? Adrenalectomy is followed by thymic hypertrophy, but the hormone content of this big thymus decreases [ 124, 137]. Administered in large amounts, cortisol induces a wave of pyknoses in the thymus and to a lesser extent in the lymph nodes [see 147]. Small lymphocytes were primarily killed by this method [ 182, 151 ]. Added to the medium, cortisol (0.01 /~g/ml) or corticosterone (0.1 ~g/ml) shortened the survival time of lymphocytes [291,462, 519]. At 2.0/~g/ml cortisol killed the small lymphocytes from embryonic mouse thymus. At 50 ug/ml it killed all cells [430]. The surviving cells show an increased helper potency [106] and an increased potency to induce the graft-vs- host reaction [65, 107]. Following a single injection of 2 mg of cortisol the spontaneous Thy 1 positive, rosette-forming cells did not decrease in number, while total lymphocyte count decreased by 90% [33]. In conclusion, cortisol killed the immature cells only. This observation was confirmed over and over again. Cortisol appeared to kill other cells, besides the small (immature) thymic lymphocytes. In mice injected with 15 mg of cortisol, the mono- nuclear cells disappeared from the blood [497]. Three hundred micrograms of cortisone/g body wt. were injected into mice. Spleen cells from these mice did not differentiate into cytotoxic cells when incubated with allogenic spleen cells, however, they developed helper cell function. It was concluded that another member of the cell-to-cell interaction was killed [316]. Speculatively this cell was identified as the macrophage. Spleen cells from mice, stressed by ether anesthesia, produced few plaque-forming cells with sheep erythrocytes. Addition of syngeneic T cells did not result in any increase in their number, whereas a significant increase in plaque-forming cells was observed following the addition of syngeneic B cells and macrophages. In conclusion, stress results in damaging B cells and macrophages, but not T cells [207]. It remains open to discussion as to how stress resulted in this effect. It should be noted that at weak concentrations (10- 8) cortisol induced an increased proliferative activity 150 J. Comsa et al. in incubated lymphocytes [430, 539]. Dose response curves of these corticoid influences are much needed. From these observations, it is not possible to understand the immunostimulant influences of corticoids administered in small amounts. Concerning this peculiar aspect of this phenomenon, we only know that the rejection of a second skin allograft is not influenced by adrenalectomy in rats which had already rejected a similar first graft: Rejection time (days) First Graft Second Graft Normal rats 7.1 -+ 1.3 4.3 + 0.5 Adrenalectomized 12.2 +- 1.5 4.4 -+ 0.5 Adrenalectomized and thymectomized 15.9 -+ 1.6 4.6 +- 0.5 Thus, the immunostimulant effect of the adrenal cortex appears to express an influence of corticosteroids on the differentiation of immunocompetent cells. As soon as this occurs, corticoids are no longer necessary. The influence of corticoids and of thymic hormones seems to be exerted on the same stage of the immune response.

3 The Thyroid

To our knowledge, the first observation concerning the role of the thyroid in immune response was made by Houssay and Sordelli [260]. Thyroid- ectomized rabbits and horses were injected with sheep erythrocytes, typhoid vaccine or tetanus or diphteria toxoid. They produced less anti- toxins than intact ones, whereas the agglutinin and hemolysin titers of their serum were equal to those of intact animals. Rats were thyroidectomized at birth. They were injected with sheep erythrocytes at the age of 2 months. Plaque-forming cells in their spleen decreased in comparison with intact rats. Daily thyroxine injection (10 micrograms/animal, the reason for this dose is not explained) restored the normal reaction [ 173]. Humans were injected with sheep erythrocytes or antigens of Shigella flexneri, Salmonella typhi or Escherichia coli. The antibody production was higher in Graves' disease patients and lower in hypothyroid patients than in healthy subject [501]. Guinea pigs were inoculated with tuberculosis bacilli and injected with a lethal dose of tuberculin. In intact animals this induced a rise in body temperature, but in thyroidectomized animals it did not. Serum from tuberculosis-injected guinea pigs was injected into normal animals together with tuberculin. The temperature of recipients rose if the serum donor were intact, but not if the donor were thyroidectomized [288]. Hormonal Coordination of the Immune Response 151

One-month-old male Sprague-Dawley rats received DA skin grafts. The recipients were thyroidectomized, thyroidectomized and thymectomized, thyroidectomized and adrenalectomized or subjected to all three operations. Thyroidectomy by itself delayed graft rejection (7.1 -+ 1.1 days - 9.7 -+ 1.3 days). In thymectomized, adrenalectomized and thymo-adrenalectomized rats the rejection (delayed already by these operations) was not further delayed by thyroidectomy. Daily thyroxine injections (5 ~g/ 100 g, the dosage known to be sufficient to prevent thiouracil goiter) restored the normal rejection in thyroidectomized rats, but had no influence on thymectomized, adrenalectomized or thymo-adrenalectomized [ 118]. Thus, at least in the peculiar case of graft immunity: 1. The immune response is depressed by thyroidectomy and restored to normal by thyroxine in thyroidectomized animals. 2. Neither thyroidectomy nor thyroxine influences the immune response in the absence of the thymus or the adrenals. The influence of the thyroid on the immune response appears to be mediated by the thymus and the adrenal. (Both thymus and adrenal are atrophic following thyroidectomy, see Sect. 6.2.2-6.2.4.)

4 The Gonads

Female mice are better humoral immune responders than males [e.g. 46, 215, 477]. F (C 57bl × BALB) mice were injected with erythrocytes from BPS mice. Their serum was fractionated in order to separate 7S and 19S antibodies. In males hemagglutinins were present in the 19S fraction only, whereas in females both fractions contained hemagglutinin [45]. Skin allograft rejection was faster in females than in males [90, 238, 472]. Castration was followed by an accelerated allograft rejection in males, less so in females [90, 238, 472]. Syngeneic grafts of ovaries in males and of testes in females had no significant influence on allograft rejection [90, 238, 4721. Testosterone injections (650 ~g three times weekly) delayed the rejection in castrates [89]. In rats similar results were observed [ 119], the production of hemagglutinins against allogeneic mouse erythrocytes increased in castrates but hemagglutinin production against sheep erythrocytes decreased. Estradiol injections in male mice were followed by an increased antibody production (e.g. 2.5 ug daily [ 287], 10/~g [477] ). Allograft rejection was delayed following estradiol injection (50/~g three times weekly) in X-irradiated mice reconstituted with syngeneic bone marrow [496]. The development of adjuvant arthri- tis (by injection of Freund's adjuvant) or autoimmune thyroiditis (by injection of thyreoglobulin) was delayed in female rats by daily injections 152 J. Comsa et al. of 0.5 mg estradiol and in males by daily injections of 0.5 mg testosterone [279]. In F (C 57bl × BALB) mice injected with BP8 erythrocytes, hemagglutinin production was increased in castrates and was depressed in castrates bearing estradiol implants (the daily resorption rate was not determined [45]). Skin allograft rejection was delayed by estradiol injection (500 ~g daily) in rabbits bearing a corneal graft from the skin donor strain [583]. Thus, the enhancement of immune response by estradiol was not generally confirmend. Thymectomized male rats were injected with estradiol (200 #g daily for 16 days). Their serum was added to the incubation medium of human lymphocytes. These ceils produced less rosettes with sheep erythrocytes than those incubated with normal rat serum [578]. Castrated Sprague-Dawley rats (males and females) rejected DA skin allografts in 5-6 days (intact animals in 7.1 -+ 1.0 days). Implants of estradiol in females (daily resorption rate 0.4 ~g) and of testosterone in males (daily resorption rate 40 /~g) restored the normal rejection. In thymectomized castrates of both sexes, the grafts were rejected as in thymectomized rats (9.8 - 1.6 days) and sex hormone implants were of no influence. In thymectomized castrates daily injections of the Bernardi- Cornsa thymic hormone preparation (100/~g) was followed by rejection within normal time limits, whereas the same injections were of no influence on those castrated alone. Thus, in this experiment the influence of the gonads on allograft rejection appeared to express the influence of the gonads on the thymus [ 119]. Receptors for estradiol were demonstrated on the surface [577] and in the cytosol [568] of thymocytes. Female thymectomized CBA mice were mated with T6 T6 males. They rejected T6 T6 skin grafts in normal time limits. This is not due to sex hormones. Pseudopregnancy had no similar effect. The extirpation of the fetuses on the 10th day of pregnancy (placenta left in place) resulted in a significant delay in the graft rejection which can only be explained by the passage of hormones from the fetus to the mother [and not by passage of cells from the F (CBA X T6 T6) fetus] [394]. In female nude mice mated with CBA males the pregnancy did not enhance the immune response against sheep erythro cytes [ 570]. Progesterone injections had no effect on skin allograft rejection in normal mice and rabbits [e.g. 351]. In spayed female rats bearing implants of progesterone (they resorbed 400 ug daily of the steroid), skin allografts were rejected in 9.3 +- 0.9 days, whereas spayed controls did so in 5.8 -+ 1.0 days. In spayed females bearing similar progesterone implants associated with estradiol implants (they resorbed daily 0.4 #g of the latter), skin allografts were rejected within 7.6 -+ 1.1 days. In other words, these rats reacted like normal females (rejection within 7.1 -+ 0.8 days) and not like castrates. Estradiol + progesterone -- 1:1000 is the well-known Hormonal Coordination of the Immune Response 153 ratio of these hormones which induces a pseudopregnancy in spayed females. Administration of estradiol and progesterone under these conditions was thus observed to suppress the consequences of ovariectomy on the immune response as well [119]. Spleen cells of pregnant mice showed a decreased activity in mixed lymphocyte culture but an increased plaque- forming cell activity. These modifications could be reproduced with injections of human chorionic gonadotropin but not of progesterone. Osoba observed that [3941 (see above): 1. Pregnant thymectomized mice reject skin grafts like normal animals. 2. Extirpation of the fetuses but not of the placentas results in a significant delay in rejection. 3. Pseudopregnancy has no similar consequences. These observations seem to exclude the influence of the corpus lute- urn and to emphasize the influence of the placenta on the immune response. An immunostimulant influence seems to emanate from the fetus (perhaps from its thymus). These observations allude to the controversial problem of the mother-fetus tolerance. Mating between animals histoincompatible to each other results in a normal pregnancy and the offspring are reared normally. Immunosuppressive influences seem, therefore, to be active during pregnancy. Experimental analysis of this phenomenon is scarce. The attempts to reproduce the influence of pregnancy with hormone injections were not always performed under valid conditions, that is: In castrated animals Parallel experiments with injections of progesterone alone and with progesterone associated with estradiol in the well-known ratio of 1000: 1, which induces the pseudopregnancy reaction in the gonaducts of ovar- ectomized animals. According to Osoba an immunostimulant influence seems to emanate from the fetus itself and the placenta seems to exert an inhibitory influence. In fact, the role of the placenta is a matter for future research. The placenta is a plurivalent endocrine gland. The list of the hormones produced by the placenta is not yet complete, but an immunosuppressive influence may emanate from estriol, which the placenta produces in large amounts.

5 The Adenohypophysis

The description of the consequences of hypophysectomy on the immune response are divergent from one author to another. Hypophysectomized mice were injected with sheep erythrocytes but few plaque-forming cells 154 J. Comsa et al. developed in their spleen. The normal reaction was restored by injections of growth hormone or corticotropin [209, 210]. Plaque-forming cells and serum agglutinin titers were significantly low in hypophysectomized rats injected with sheep erythrocytes [397]. Hypophysectomized Sprague- Dawley rats grafted with DA rat skin rejected those grafts with a significant delay [123]. Hypophysectomized Wistar-Furth rats produced fewer antibodies against sheep erythrocytes and rejected skin allografts later than normal rats [575]. Divergent observations were registered too. Hypophysectomized rats reacted normally to sheep erythrocyte injections [ 159,277]. We cannot explain these divergencies. The consequences of hypophysectomy on immune response were revealed only after a subsequent X-irradiation [502]. In these animals, both skin allograft rejection and hemagglutinin production after sheep erythrocyte injection were significantly depressed [277]. The observations on immune response in Snell-Bagg dwarf mice were explained. These mice are dwarves inasmuch as they suffer from a severe adenohypophyseal deficiency (see Sect. 6.2.2). Dwarf mice are severely immunodeficient. Following sheep erythrocyte injection they develop significantly few plaque-forming cells in the spleen [36, 37, 277]. Serum agglutinin titers were low after sheep erythrocyte or Brucella injections [157]. Dwarf mice did not reject allografts [175]. The secondary immune response of dwarf mice to sheep erythrocytes was normal [38]. Neverthe- less, thymocytes or spleen cells from dwarf mice induced the in vivo graft-vs-host reaction if injected into F (SB × AKR) mice [ 158].

6 Influence of Hypophyseal Hormones on the Immune Response

6.1 Growth Hormone

Growth hormone injections in hypophysectomized mice were followed by restoration of the immune response [209, 210]. Growth hormone injection in dwarf mice was followed by activation of the immune response [e.g. 175, 397]. Thyreotropin and thyroxine had a similar effect [37]. The enhancement of the immune response (number of plaque-forming cells in the spleen after injection of sheep erythrocytes) was particularly significant when growth hormone and thyroxine were injected together [413]. The thymus is involved in this effect of growth hormone. In thymectomized dwarf mice, growth hormone injections had no effect on the immune response, but they induced normal growth [174]. This involvement of the thymus in the influence of the growth hormone on the immune response was observed in hypophysectomized rats. In hypophys- Hormonal Coordination of the Immune Response 155 ectomized and thymectomized rats the dose-response ratio of growth hormone to growth was low although some effect was observed [see 329 for review]. However, the growth hormone injections had no effect on the immune response of these rats. Hypophysectomized and thymectomized rats produced no antibodies against human serum albumin. The normal response was restored by injections of growth hormone and the Bernardi-Comsa thymic preparation, not by growth hormone or thymic hormone alone [125]. Skin allograft rejection was delayed to the same extent in hypophysectomized and in hypophysectomized thymectomized rats. In those only hypophysectomized, the normal reaction was restored by growth hormone injections; in those thymectomized and hypophysectomized, growth hormone and thymic hormone injections had the same effect (but not growth hormone or thymic hormone alone [ 123]). These observations may explain the influence of thyreotropin or thyroxine on the immune response of dwarf mice. Thyroxine is a permissive factor for the secretion of thymic hormone [ 124] and indeed thyroxine injections enhanced the immune response in dwarf mice, but not in neonatally thymectomized mice [ 16] of normal strains.

6.2 Corticotropin

The humoral immune response was depressed by corticotropin injections. Antibody production against ovalbumin [190], pneumococcal antigen [203, 621, horse serum [250], typhoid vaccine [373], and bovine serum albumin [341,389] was severely inhibited following corticotropin injection in large doses. There were remarkably few plaque-forming cells in the spleen of intact mice when these were injected with corticotropin and sheep erythrocytes. On the allograft immune response corticotropin influence was less obvious. A slight delay was observed in mice [3511 and no influence was observed on rhesus monkeys [305], rabbits [9], guinea pigs, pigs or humans [ 170]. These experiments were carried out on intact animals. The corticotropin dosage is difficult to understand, since corticotropin doses were expressed in units of weight from the start, regardless of the successive purifications. In hypophysectomized and thymectomized rats, daily injections of 300 milliunits of corticotropin were followed by a remarkably accelerated skin allograft rejection (17.8 -+ 1.2 days - 6.7 -+ 0.8 days). Additional injections of the Bernardi-Comsa thymic hormone preparation resulted in a nearly complete suppression of the corticotropin effect (rejection in 15.2 -+ 1.2 days) [ 123]. From previous experiments it was known that thymic hormone is a synergist to growth hormone and that it antagonizes 156 J. Comsa et al. corticotropin. The immunological tests mentioned above gave results consistent with this notion. There is little to be said about the influence of other adenohypophyseal hormones on the immune response. The restoration of a normal response to sheep erythrocytes with thyreotropin injections [175] has been mentioned already. In hypophysectomized and thymectomized rats thyreotropin injections had no influence on skin allograft rejection (J. Comsa and H. Leonhardt, unpublished observations). Thus, the influence of thyreotropin may be mediated by the thymus via the thyroid, see Sect. 3. Pancreatectomy was followed in rats by a delayed rejection of skin allografts and no change in the immune response to sheep erythrocytes, Brucella antigen or bovine serum albumin. As far as we know, this is the first attempt to investigate the influence of the pancreas on immune functions.

7 Mechanisms of Hormonal Influence on the Immune Response

This problem can be considered from two aspects: 1. Events on the cellular of subcellular level which may enable us to understand the hormonal influences. 2. All the hormones mentioned above are present in normal animals. Intricate interactions between them were demonstrated by several tests. To what extent could these interactions influence the immune response? Can we imagine a hormonal coordinating mechanism for immune functions?

7.1 Cellular and Subcellular Aspects of Hormonal Action

The following summary is by far inexhaustive. This would unreasonably exceed the limits of this review.

7,1.1 The Corticosteroids

Corticoids enter the cells, are fixed to a transcortin analog in the cytosol [e.g. 322,383,497; see 187], traverse the cell thus bound, and are finally fixed to specific receptors in the nucleus [e.g. 449, 466, 546]. This is completed within minutes [466]. The nuclear receptor was thought to be a histone [467] or an acidic protein [e.g. 2]. The binding protein is perhaps essential for corticoid action within the nucleus. Purified "chroma- tin" is able to induce RNA synthesis in vitro. This synthesis is depressed by protein-bound cortisol, but not by free steroids [446]. The presence Hormonal Coordination of the Immune Response 157 of specific corticoid receptors on lymphocytes is essential for the cytotoxic corticoid effect. Incubated with cortisol, the corticoid receptors of lymphocytes decrease with time in the surviving cells [49]. (The cells bearing a dense receptor net are killed.) Corticoids increase the stability of cellular and subcellular membranes. Hemolysis by antibodies or by physiochemical agents is inhibited by corticosteroids [e.g. 4, 272]. Hamster kidney cells infected with rubella virus are not lysed by antirubella serum if cortisol is added to the medium [498]. Intracellularmembranes were also found to be stabilized by adding cortisol [ 162]. Cortisol inhibited the release of lysosomal enzymes into the cytosol [532, 534]. Corticoids interfere with glucose metabolism. Glucose transport and phosphorylation [69, 3831, glucose uptake [281, 375], and ATP synthesis [391] are inhibited by adding cortisol (at 10 -7- 10 -6 M concentration). Metabolism by the pentose cycle was found to be enhanced [69], this action is blocked, however, by puromycin [376,339]. It was concluded that cortisol induces the synthesis of a proteidic inhibitor of glucose metabolism. Corticoids inhibit DNA and RNA synthesis [see 495 for review]. Thy- midine uptake [339, 340, 376], DNA polymerase [290], and thymidine- kinase activity [241] are depressed, also uridine uptake [199, 551], adenine uptake [77], RNA polymerase activity [2, 68, 194, 340], and DNAse activitiy is enhanced [443]. Corticoids inhibit protein synthesis in most tissues (except in the liver). Amino acid uptake [e.g. 64, 181,227, 375,537] is depressed in presence of cortisol and so is the activity of various transaminases [e.g. 64]. The inhibition of lymphokine synthesis [ 523] is particularly relevant to our topic. Amino acid release by incubated lymphocytes is enhanced in presence of cortisol [489]. Thus, cortisol appears to inhibit the anabolism and to promote the catabolism of nucleic acids and proteins. These effects were obtained with cortisol at high concentrations ( 10 -7 - 10 -6 M). They are supposed to be direct with no second messenger needed [495]. Yet cortisol injections depressed the adenylate-cyclase activity in X-irradiated rats [285]. It is interesting to note that the stimulating effect of cortisol at low concentrations on lymphocyte proliferation in vitro (10-a M [ 539] ) was supposed to be mediated by cyclic AMP. It should also be noted that the stabilizing effect on lysosome membranes in vitro was observed at low, but still immunostimulant concentrations [ 144]. These two observations are the only ones possibly related to the immunostimulant influence of corticoids within the physiological range. 158 J. Comsa et al.

7.1.2 Peptidic Hormones It is generally accepted that peptidic hormones do not enter the cell. Thus a "second messenger", transporting the hormonal message from the cell membrane (to the nucleus?), becomes relevant.

7.1.3 Cyclic Adenosine Monophosphate (cAMP) Added to the medium, cyclic AMP stimulates lymphocyte proliferation [333]. Injection of sheep erythrocytes into immunized mice resulted in an increase in the cyclic AMP level in their spleen lymphocytes within 2-10 min [418]. When (butyryl)- cyclic AMP was added to the medium [e.g. 70, 266, 530], plaque-forming cells increased in number in spleen cells from mice immunized against sheep erythrocytes. Adenylcyclase stimulating catecholamines (e.g., epinephrine) enhanced the immune response [70] and so did those drugs which inhibit phosphodiesterase [72]. This is, however, only valid up to a certain point. When added in excessive amounts, cyclic AMP inhibited plaque-forming cell differentiation [73]. The cytolytic activity of immunized C57 b 1 mouse spleen cells against DBA mastocytoma was inhibited by cyclid AMP [254]. Plaque- forming cell formation was inhibited in mouse spleen cells by cyclic AMP added to the medium. (Was it added in excess in these two experiments?) Rosette-forming cells increased in number in human lymphocytes by administration of drugs which increase the cyclic MP content of lymphocytes and then decreased when dibutyryl-cyclic AMP was added to the medium [99] (perhaps in excess, see above).

7.1.4 Synthetic Polyribonucleotides The enhancing effect of poly (A:U) on the immune response [ 56, 72, 74, 73, 266, 343] at low concentrations (high concentrations show an opposite effect [72]) was interpreted as an enhancing influence on cyclic AMP synthesis [70]. Poly (A:U) injected into neonatally thymectomized mice induces a normal rosette-forming cell differentiation and a normal allograft rejection [126]. Still poly (A:U) added to the medium enhanced cytotoxic activity of spleen cells cultivated with allogenic cells in normal mice but not in the nude ones (the thymus was necessary [56]). Prosta- glandins were also said to enhance the immune response [265] and the prostaglandin synthesis inhibitors did as well [531 ]. In this new field the observations are still confusing. Poly (A:U), prostaglandin E2 or cyclic AMP, added to the medium, induced the potency of graft-vs-host reaction in spleen cells from thymectomized mice; incubation of mouse lymphocytes with thymic humoral factor resulted in an increased cyclic AMP content [299]. Incubation of Hormonal Coordination of the Immune Response 159 spleen cells from nude mice with thymopoietin, cyclic AMP or poly (A:U) induced the appearance of antigenic markers and helper function [451]. Neither thymosin [20] nor serum thymic factor [20] induced an increased cyclic AMP content in thymocytes when added to the medium. Also, incubation of thymocytes with serum from normal or thymectomized pigs induced an increased cyclic AMP content in these cells, whereas the serum thymic factor produced by synthesis did not [34]. Perhaps, this partly confusing picture could be somewhat clarified if we knew the consequences of thymectomy on the cyclic AMP level of lymphocytes. Remarkable as it may sound, we do not.

7.2 Hormonal Interactions

The hormones mentioned thus far are simultaneously present in the organism. The hormonal influence on immune response (i.e., its action directly on the immune system or mediated by another endocrine) is expressed by an algebraic sum. This assertion is self-evident. Thus far, the documentation concerning this topic is poor. Elements of the hormonal interactions in general must be noted. (Observations mentioned without references are described in extenso [ 114]). Hormonal interactions must be seen from two different aspects: 1. The reciprocal influence between gland A and gland B 2. The interaction between the hormones of gland A and gland B in their circulating form These two aspects probably are linked to each other in some way. However, we can only assume this up to now.

7.2.1 Consequences of Thymectorny on the Endocrines Following thymectomy, a transitory stimulation of the thyroid, and the gonads, and the adrenal cortex was observed in guinea pigs and rats [ 114]. These endocrines returned to normal in rats and in those guinea pigs which recovered from thymectomy. In those which waste (examined sub finem) degenerative changes were observed. This was the case in thymectomized mice. As far as we know, the endocrines were examined in wasted mice only [ 155]. This is insufficient. Degenerative changes in the endocrines occur in wasted animals, whatever the reason for wasting may be (e.g., in the terminal stage of deficiency diseases). The consequences of thymectomy on the adrenal cortex seem ambiguous at first sight. Evi- dence of stimulation was noted [see 114, 140]. However, the corticosteroid content of the systemic blood was found to be decreased [ 137, 177] whereas aldosterone content was increased [ 177]. Still, the corticosterone 160 J. Comsa et al. level increased in the efferent blood of the adrenal [79,470]. This discrepancy is believed to result from an accelerated denaturation of corticosterone in thymectomized animals, perhaps mediated by the stimulated thyroid. Indeed, thyroxine injections were followed by an accelerated reduction of ring A in steroid molecules [550]. A methodical investigation of the steroid turnover rate in thymectomized animals would be of interest. In the adenohypophysis, thymectomy was followed by signs of stimulation of, more or less, all cells. The sequence of the events is different from one species to another. In rats [114, 1371 and in mice [55], the stimulation of ~ cells occurs early. In guinea pigs [ 114], 6 cells precede the ~ cells, which show signs of stimulation only in recovering animals. In wasted guinea pigs the adenohypophysis is completely dedifferentiated (consisting only of small chromophobes). In mice, the stimulation of cells appears before the onset of wasting [415]. In thymectomized axenic mice, which do not waste following thymectomy, similar changes in the adenohypophyseal cells occurred [415, 475]. Further research concentrated on the ~ cells. Electron microscopy indicated an enhanced endoplasmic reticulum and an almost total disappearance of secretion grains [562, 475] in thymectomized mice. This evidence of an increased activity is consistent with the increase in the growth hormone level in the serum [ 137]. We noted that following thymectomy the efficiency of the growth hormone is decreased (by the loss of its synergist). This results in an increased growth hormone secretion by a negative feedback effect. All these changes were prevented by thymic grafts in mice [54] or by daily injections of the Bernardi-Comsa thymic hormone preparation [see 113]. The increase in the serum growth hormone level following thymectomy was prevented by injections of the same compound [562]. The nude mouse is supposed to suffer from a severe thymic deficiency. But thymosin could be prepared from the rudimentary thymus of nude mice [573]. The histological picture of the endocrines shows to a certain extent that of thymectomized mice at the terminal wasting stage. The thyroid shows degenerative changes. In the adrenal at the age of 50 days, the reticular zone of the adrenal cortex is peculiarly large. At 90 days the cortex is shrunken. The growth hormone cells of the adenohypophysis show the same picture as in thymectomized mice. Blood thyroxine levels are low. Corticosterone levels temporarily increased (at 14 days) and later decreased. Sex steroids decreased. In vitro, adrenals of nude mice produce less corticosterone, more desoxycorticosterone, and as much aldosterone as those of normal mice. Addition of corticotropin to the medium results in normalization of the steroids produced [408, 409]. Thymic grafts in nude mice suppressed all degenerative changes in the Hormonal Coordination of the Immune Response 161 endocrines. The consequences of thymic extract injection on the endocrines of nude mice have not been investigated as far as we know.

7.2.2 Thymus-Thyroid Following thyroidectomy, the thymus undergoes atrophy of the senile type in infant guinea pigs [see 114]. The hormone content of the thymus, the lymph nodes, and the spleen decreased in thyroidectomized rats. This could be prevented by daily injections of 5/~g of thyroxine, the minimal dose for preventing thiouracil goiter [ 124]. In guinea pigs, the minimum dose necessary was 2 /~g daily. Increased thyroxine amounts induced thymic hypertrophy (up to 16 ~g). Larger amounts, however, resulted in a relative atrophy with histological signs of intense stimulation, which points to the picture of human thymus in Graves' disease. The lymphocytosis induced by thyroxine injections is mediated by the thymus. In doses inducing lymphocytosis in normal guinea pigs, thyroxine injections are followed by lymphopenia in thymectomized animals [114]. The thyroid seems to regulate in some way the thymus functions. Thymic hypertrophy induced by castration is inhibited by simultaneous thyroidectomy [344] as well as an increase in the hormone content of the thymus in castrates [ 124]. The influence of the thyroid on the thymus is probably direct. Thymic hormones and thyroxine are mutual antagonists (demonstrated with the Bernardi-Comsa compound [see 1141 and with thymosin [see 127]. The influence of the thymus on the thyroid (the thyroid stimulation following thymectomy) is more intricate. The thymus antagonizes thyreotropin and it decreases the thyreotropin secretion [see 114].

7.2.3 Thymus-Adrenal The influence of the adrenal on the thymocytes was described above. In addition, an adrenal influence on the thymic glandular epithelium seems probable. Adrenalectomy is followed by thymic hypertrophy [see 114 for review], but the hormone content of this enlarged thymus is low [ 124, 137] and increased above the normal level in adrenalectomized rats with corticosterone or desoxycorticosterone [ 124]. The influence of the thymus on the adrenal cortex is intricate. The stimulated condition of the adrenal cortex following thymectomy (prevented by thymic hormone injections) was mentioned above. The adrenal of thymectomized rats was incubated in vitro. Addition of thymosin to the medium was followed by an increased oxygen consumption. The oxygen consumption of adrenals from thymectomized mice previously injected with thymosin or with homeostatic thymic hormone was low 162 J. Cornsa et al.

(equal to that of normal mice if homeostatic thymic hormone was injected, but still 30% higher after thymosin injections [140] ). Obviously, two mechanisms interplay: (a) the antagonistic action of the thymus toward corticotropin (see above) and (b) a possible direct action of the thymus on the adrenal cortex. The thyroid seems to play a permitting role on the adrenal's influence on the thymus. The "big empty thymus" of adrenalectomized rats is not seen in tyhroidectomized and adrenalectomized rats. In these rats, the thymus shrinks just as in those thyroidectomized alone.

7.2.4 Thymus-Adenohypophysis

Following hypophysectomy the thymus shrinks, and the picture is that of senile involution. The lobules shrink, the cortex is narrow but particularly well delimited from the medulla, there are no lymphocytes in the medulla, and the lymph nodes shrink [452]. Thymidine uptake is decreased in the thymus and in the spleen [293]. The validity of this test could be questioned because hypophysectomy had no effect on thymidine uptake in the testes [293]. Antigrowth hormone antiserum induced a similar thymic atrophy [411]. The hormone content of the thymus decreased as well as the stores of thymic hormone [124, 128, 137] in the lymph nodes and the spleen. Daily injections of growth hormone, corticotropin or thyreotropin prevented these changes [ 124]. The shrunken lymph nodes of hypophysectomized rats are restored to normal by growth hormone injections. These observations may demonstrate: 1. An influence of growth hormone on thymic lymphocytes, this is an acquired characteristic. There are specific receptors for growth hormone on the surface of lymphocytes [e.g. 397]. Im lymphocyte cultures growth hormone addition is followed by an increased mitosis rate [333], an increased thymidine and uridine uptake [397,490]. This action is supposed to be mediated by cyclic AMP since addition of coffeine to the medium decreased the minimal efficient growth hormone concentration by two-thirds [333]. Growth hormone seemed to favor the maturation of thymocytes. Thymectomized mice, injected with sheep erythrocytes, develop few plaque-forming cells. Injection of growth hormone and thymocytes was followed by an increase in the plaque-forming cell number, whereas neither growth hormone alone, nor thymocytes alone had any effect [ 16]. Injection of newborn Long-Evans rat spleen cells in newborn Ch River rats (of Sprague-Dawley origin) induced runting syndrome only when injected together with growth hormone. The influence of growth hormone thus appears to be exerted on Stutman's postthymic immature cells. Hormonal Coordination of the Immune Response 163

2. An influence of growth hormone on the secretory thymic reticulo- epithelium. This ir purely hypothetical. It has also been documented that growth hormone is a synergistic to thymic hormone (see above). A clear-cut difference appears between the stimulatory influence of growth hormone on the thymus and the synergism between growth hormone on the thymus and the synergism between growth hormone and thymic hormone. It has been mentioned already that corticotropin injections restored the hormone content of the thymus, the lymph noces, and the spleen in hypophysectomized rats. The mechanism of this phenomenon is open to discussion. It can be understood as being mediated by the adrenal cortex (see above) but a direct influence of corticotropin on the isolated rat thymus has also been observed [137]. The restoration of the hormone content in the thymus of hypophysectomized rats by thyreotropin can certainly be understood as being mediated by the thyroid. Injections of an antigrowth hormone antiserum in 27-day-old mice resulted in a wasting syndrome similar to that induced by neonatal thymectomy. The thymus of these animals shrunk and their immune response became deficient. In their hypophyses the growth-hormone cells were modified as in thymectomized mice. Their thymuses shrunk [475]. Injec- tions of an antithymus antiserum had no similar effect, perhaps because it was principally an antithymocyte serum. For this test, antithymic anti- sera should be prepared with cells from the secretory thymic reticuloepithelial cells. The dwarf mouse suffers from a severe adenohypophyseal deficiency [e.g. 40, 321, 471]. This deficiency is conditioned by the adenohypophysis itself and not by the hypothalamus. The graft of a syngeneic normal hypophysis in the sella turcica of dwarf mice was followed by normal growth [88]. Substitutive therapy with adenohypophyseal and other hormones to some extent produced confusing results. Dwarf mice resumed growth when injected with growth hormone, thyreotropin [47] or thyroxine [236, 390, 472]. Bioassay and polyacrylamide electrophoresis of a crude hypophyseal extract of dwarf mice demonstrated a severe growth hromone deficiency [321 ]. Thus, dwarf mice are dwarves inasmuch as they are deficient in growth hormone (in fact, the other hypophyseal hormones have not been researched as far as we know). The thymus, the lymph nodes, and the splenic white pulp are atrophic in dwarf mice. At 20 days the thymus shows senile atrophy, and at 60 days the terminal stage of deficiency diseases [36]. Yet this thymus was proven functional (see Sect. 5). Thyroid and adrenals are atrophic. The restoration of dwarf mice by thyreotropin or thyroxine injections raises some problems. The restoration of the immune response by thyroxine 164 J. Comsa et al. may result from the influence of thyroxine on the thymus. We know, indeed, that thyreotropin restored the thymus function in hypophysectomized rats. Unfortunately, we do not know which changes in the thymus of dwarf mice are induced by thyroxine. In any case, this explanation is unsatisfactory since thymic hormone was shown to be of no influence on the immune response in hypophysectomized animals, unless growth horrhone had also been injected (see above). Thus, we could sus- spect that thyroxine injections in dwarf mice induced an increased growth hormone secretion also. It is, indeed, open tO discussion whether the adenohypophysis of dwarf mice is asleep or dead. In the first case, thyroxine may induce a chain of events in the adenohypophysis of thyroidectomized rats, the growth hormone content tending toward zero. It is restored with thyroxine injections [321]. There is circumstantial evidence that the well-known hypertrophy of the adrenal cortex following thyroxine injections is mediated by the adenohypophysis [e.g. 164]. Corticotropin injections resulted in an increased growth hormone secretion [e.g. 480, 555]. In dwarf mice growth hormone injections showed a gonadotropic effect. Puberty was induced and fertilization, pregnancy, and breeding were possible [39]. An intricate interaction within the cells of the adenohypophysis could be possible in the particular case of the dwarf mouse which is injected with one or another hypophyseal hormone. This presumption indicates that the adenohypophysis of the dwarf mouse is only in a deep resting state (asleep) and is still able to respond to stimulation. In comparing this hypophysis-to-hypophysis interaction, could a thymus-to-thymus interaction be acquired? The thymus also secretes several hormones. The answer is short and simple: We just do not know. Thus, an intricate network of hormonal interaction appears to influence the immune functions. The notion of a coherent coordinative mechanism for these functions can be imagined. This suspicion could be supported further from the observations made on the nervous system's involvement in these interactions. Destruction of hypothalamic areas was followed by a depression of the immune response; their stimulation by faradization enhanced it [294]. Serum corticosterone level, action potential of the hypothalamic ventromedian area, and the number of plaque- forming cells in the spleen increased in demonstratively parallel curves following sheep erythrocyte injections in rats [54]. The stimulatory influence of antigens on these mechanisms seems to be exerted at different levels and not on the hypothalamus alone. Corticosterone production by incubated adrenals was increased, following addition of an antigen (enterotoxin) to the medium [224], and the corticotropin release of incubated hypophyses was also increased by the addition of E. coli endotoxin [491,358]. Hormonal Coordination of the Immune Response 165

The thymus may exert an influence on this mechanism. RNA synthesis increased in the hypothalamus of thymectomized rats incubated in vitro. This was prevented by thymosin or homeostatic thymic hormone injections. Added to the medium, both hormones caused a decrease in RNA synthesis in the incubated hypothalamus of intact rats [ 142]. This seems to indicate a negative feedback effect of thymectomy on the hypothalamus. This entirely new field awaits further research.

8 Possible Alimentary Influences

The picture of coordinatory mechanisms of immune functions is not complete if alimentary factors are not taken into account. The experimental documentation on this is poor. However, a superabundant number of clinical documentations are available concerning immune disturbances in underfed human subjects. Avitaminosis A, pantothenic acid, and pyridoxal deficiency in rats and avitaminosis C in guinea pigs resulted in a decreased antibody production. Thiamine, riboflavine, and folic acid deficiency were of no influence [see review 23]. Pair-fed controls showed a normal immune response, however, their thymus was also atrophic. Thus, this influence of specific deficiencies on the immune response did not result from the underfeeding which is by definition a consequence of avitaminoses. Rat fed a pyridoxine-deficient diet had no immunocompetent mature T cells in the thymus. Thymocytes from these rats acquired immunocompetence when incubated on a monolayer of thymic reticulo- epithelium from normal rats, but not if the reticuloepithelial cells were taken from pyridoxine-deficient rats [540]. This suggests that pyridoxine deficiency may interfere with the immune response inasmuch as it inhibits thymic hormone production. This is perhaps not the only way. Lympho- cytes from BALB mice incubated with irradiated C3H lymphocytes developed cytotoxic functions, which decreased in time after the antigenic lmyphocytes were added after more than 20 days of culture. After 35 days of culture, the ability to develop cytotoxic functions was lost. It was restored by addition of pyridoxal to the medium [464]. Here again a new field of interest opens. As long as our documentation on this subject remains as poor as it is, the coordinatory mechanisms of immune functions will not be completely understood. 166 J. Comsa et al.

9 General Conclusions

This review contains evidence for the influence of nearly all hitherto known hormones on the immune response. As stated above, the documentation for this evidence differs quantitatively from one hormone to another: abundant for the thymus, in general it is scarce for the other endocrines, because the attention of immunologists has thus far focused more or less exclusively on the thymus. This is unjustifiable. There is valid evidence for pluriglandular influences on the immune response. Korneva's observations indicate an influence of the hypothalamus [294]. It was postulated for the thymus, that its influence is needed for the differentiation of the immune system (thymectomy is of no consequences in animals whose immune system is already mature). This is not entirely valid. Endocrine mutilation resulted in immune disturbances in prepuberal animals, as wag shown above. A difference mfist be made between mature lymphocytes and senti- tized lymphocytes. In the lymphocytes, the first contact with an antigen results in changes which are under hormonal influences. Once these changes have occurred, hormones are apparently no longer needed (the secondary immune response is not influenced by endocrine mutilation. The mechanism for these hormonal influences differs from one endocrine to another. Some act directly on the immune system. The synergic growth hormone, the thymus, and the corticosteroids are examples of this condition. Some endocrines influence the immune system inasmuch as they stimulate or inhibit other glands. This may occur in two different ways: 1. An interaction between two hormones in their circulating form (such as the interaction thymus-corticotropin) 2. An influence of a hormone on a gland itself; the influence of the thyroid and the gonads is mediated by the thymus. A scheme (Fig. 1) summarizes those interactions. Do these observations themselves justify the postulate of a hormonal coordination of the immune response? At first glance, no. It requires the demonstration of an induction of the hormonal reaction by a specific stimulant. The stimulant may act directly (the isolated perfused pancreas secretes increased amounts of insulin if the glucose content of the perfusion liquid is increased). The reaction may result from an intricate mechanism involving several organs (energetic metabolism increases in the cold, under the influence of the thyroid and the adrenal cortex, but not in hypophysectomized animals). In order to accept the postulate of a hormonal coordination of the immune response, we must observe, that the presence of an antigen within the organism results in an increased activity Hormonal Coordination of the Immune Response 167

Fig. 1. Summary of the hormonal coordi- Hypothalamus Antigen nation of the immune response. Stimulating influences, - .... //inhibiting l× influences. 1, adenohypophysis; 2, thyroid; 3, thymus; 4, gonads; 5, adrenal; 6, lymphocytes. Abbreviations: BC, thy- STH ACT'H mic hormone (Bernardi-Comsa); TS, testosterone; OF., estradiol; X, unknown; STH, growth hormone; TSH, thyreotro- , T~T3 pin; ACTH, corticotropin; T4 Ta, thyroid 3 hormone

of the endocrines mentioned above. This evidence is given partially by the observations of Besedowsky and Sorkin [54]: in presence of sheep erythrocytes there is a parallel increase of the action potentials in the hypothalamus, the corticosterone content of the blood, and the number of plaque-forming cells in the spleen. It would be of supreme interest to extend this fundamental observation to the other endocrines known to influence the immune system. Be that as it may, our knowledge of these mechanisms will remain incomplete as long as alimentary factors are not taken into account. The observations of Willis-Carr and St. Pierre are of great interest in this context [540]. Earlier documents are not entirely satisfactory, concluding (in our opinion somwhat quickly) that most alimentary factors are of little or no influence on the immune system [see 23]. This conclusion is in striking contradiction to the everyday clinical routine. It is universally known that patients suffering from malnutrition are highly sensitive to infection that can develop with a peculiarly poor reaction and a catas- trophal prognosis. This suggests the hypothesis of a state of immunodeficiency in these patients. To our knowledge, no attempt has yet been made to verify this hypothesis. On the other hand, it is known that malnutrition results in an intricate hormonal deficiency. A characteristic pattern of thymic atrophy is an early consequence of every deficiency disease. Later, the adenohypophysis is severely affected (malnutrition was called the nonsurgical hypo- 168 J. Comsa et al.

physectomy). This, in turn, affects the thyroid, the adrenal, and the gonads. The documentation reviewed allows us to postulate a link between hormonal deficiency and immune deficiency (to some extent underfed subjects suffer from immune deficiency inasmuch as they suffer from hormonal deficiencies). One may conceive that the thymus is of primary importance in this syndrome. Its atrophy is peculiarly early and pecularly severe. In dys- peptic infants underfed for therapeutic reasons the thymus may lose 90% of its normal weight and its hormone content tends toward zero [see 329]. In the late 1940s an attempt at substitutive therapy with thymic extract was made in these patients with encouraging results [see 329]. In our opinion, interesting new aspects of the physiopathology of the immune response may appear if: 1. The pluriglandular coordination of the immune response were to be considered (instead of the exclusive concentration on the thymus). 2. The accidental, acquired forms of immunodeficiency were also investigated (research has so far focused on congenital forms).

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Aarden LA, see Watson J Albuquerque EX, see Shotz- Anagnostopoulos T, see (584) 124,191 berger GS 39, 73 Bentzel CJ 84,104 Abe T, Nomura M (1) 147, Alexander E, see Auld R Ances IG, see Beitens IZ 100, 148, 168 93,103 103 Abita JP, see Romey G 11, 72 Alexander EA, see Bruns FJ Anderson B, see Blomgren H Abraham AD, Sekerin CE (2) 78,79,81,104 (65, 66) 126,149, 170 156,157,168 Alexander E, see Davidman M Anderson NC, see Narahashi T Abramson RG, see Stein RM 79, 80, 94,105 31,70 90,112 Alexander EA, see Migdal S Andreucci VE, see Herrera- Aceves J, see Diezi J 92, 106 78,110 Acosta J 80, 107 Acheson GH, see Kahn JB Alexander P, see Grant CK Andreucci VE, see Wallin JD 36, 68 (239,240) 121,177 76, 78,112 Acheson GH, see Krayer O Alexander RW, Gill JR, Anggard E, see Oliw E 97, 3, 4, 23, 69 Yamabe H, Lovenberg W, 110 Ackerman U, see Pearce JW Keiser HR 99, 103 Antoni H, see Kaufmann R 96,110 Alfonso A, see Kupchan SM 18, 68 Adam KR, Schmidt H, 4, 69 Antonio A, see Corrado AP St~mpfli R, Weig C 10, 64 Algire GH, Weaver JM, Prahn 44, 66 Adams PB (3) 119,168 RT (8) 120, 168 Aperia A, Broberger O, Thode- Adler-Graschinsky E, see Allen DG, Blinks JR 56, 64 nius K, Zetterstrom R Langer SZ 44, 69 Allen HL, Williams RD, Lovin- 99,103 Agarossi G, see Doria G (146) good CG, Ellison EH (9) Aras M, see Bank N 85, 86, 123,174 155,168 103 Agarwal K, Garby L (4) 157, Allen JMV van, see Reif AE Arbel ER, Lazzari J, Glick G 168 (429) 125,185 17, 21, 22, 49, 64 Agosier M, Christen R, Badi- Allen PA, see Tanz RD 41, 73 Archer OK, Pierce JC (12) ner O, Gasic G (5) 147,168 Allison AC, see Taylor RB 117,168 Agus ZS, Puschett JB, Senesky (494) 187 Archer P, see Good RA (234) D, Goldberg M 98,103 Allison M, Lipham E, Gott- 121,177 Ahrens RC, see Duquesnoy RJ schalk CW 100, 103 Argyris B (13) 119,168 (158) 154,174 Almeida AP, see Langer SZ Arman CG van, see Scriabine Aisenberg AC, Davis C (6) 168 44, 69 A 49,72 Aisenberg AC, Wilkes B (7) Alsen C, B6ress L, Fischer K, Axnason BG, Jankovic BD, 133, 134,168 Proppe D, Reinberg T, Waksman BH, Wennersten C Akera T, Brody TM 56, 64 Sattler RW 46, 64 (14) 117,168 Akera T, Ku DD, Frank M, Alter B J, see Sopori ML (474) Arnason BG, Vaux de St Cyr C, Brody TM, Iwasa J 42, 43, 64 124,186 ShafnerJB (15) 117,169 Akera T, see Ku DD 42, 43, 69 Althage A, see Zinkernagl RM Arnason BG, see Waksman BH A1-Adra AR, Pilanke LM (559) (588) 131,132,191 (525) 117,188 122, 190 Altmann LC, see Wahl SJ Arnaud CD, see Schneider EG Albert PF, see Hays EF (252) (523) 157,188 98,111 117, 134,178 Amarat L, see Werthamer S Arrenbrecht S, Sorkin E (16) Albuia M, see Trainin M (512) (537) 157,189 155, 162, 169 188 Ambrose CT (10) 148, 168 Asanuma Y, Goldstein AL, Albuquerque EX, Warnick JE Amerding D, Katz DM (11) White A (17) 169 13, 64 123,137,168 Asanuma Y, see Goldstein AL Albuquerque EX, see Hogan Aramann A J, see Wara W (219) 137,176 PM 17, 37, 38, 49, 67 (528,529) 137,138,189 Aschkenasy A, Bussand A, Albuquerque EX, see Kayaalp Amstutz H, see Loor F (328) Corvazier P, Grabar P (18) SO 37, 68 126,181 146,169 194 Author Index

Ashida K, see Fujiwara M Bach JF, see Dardenne M Barr PS, see Thurman GB 12, 50, 61, 67 (128, 129) 142, 162, 173 (5O0) 187 Ashida K, see Hashimoto Y Bach JF, see Lacombe M Barratt L J, Wallin JD, Rector 6,67 (309) 143,180 FC Jr, Seldin DW 79, 80, Askonas BA, White RG (19) Bach JF, see Pleau JM (417) 103 117,169 142, 184 Barry J de, see Fosset M 14, Askonas BA, see North JR Bach MA (34) 159,169 37, 53, 54, 55, 67 (392) 183 Bach MA, see Bach JF (32) Bartels-Bernal E, Rosenberry Astaldi A, Wijemans P (20) 142, 169 TL, Daly JW 38, 64 159,169 Bach ML, see Sopori ML (474) Bartke A (39, 40) 163, 164, Astaldi A, see Astaldi G (21) 124,186 169, 170 143, 169 Badiner O, see Agosier M (5) Bartoli E, Early LE 80, 103 Astaldi A, see Krisbeek AM 147,168 Bartoli E, see Conger JD (307) 134,180 Baer M, Best PM, Reuter H 83,105 Astaldi G, Astaldi A, Groene-. 31, 37, 59, 64 Barton LJ, Lackner LH, Rector woud M, Wijemans PS, Baguerra J, see Moeschlin S FC Jr, Seldin DW 91,103 Schellekens PG, Eijsvogel (373) 146,155,182 Bartter FC, see Fill JR Jr VO (21) 143,169 Baguerra R, see Moeschlin S 99,106 Astaldi GCB, see Krisbeek AM (373) 146,155,182 Basch RS (41) 117, 1 70 (307) 134,180 Bahlmanns JS, McDonald J, Basch RS, Goldstein G Attallah AA, Lee JB 97,103 Ventom MG, Wardener HE de (42, 43) 119, 141,170 Attwell D, Cohen I, Eisner D, 96,103 Basch RS, see Sunshine GH Ohba M, Ojeda C 15, 22, 64 Baines AD 81,103 (488) 141,187 Auerbach R, Globerson A Baker PF 64 Basseches P, Bianchi CP 26, 64 (22) 117, 139,169 Baker PF, Blaustein MP, Hodg- Bassingthwaighte JB, Fry CH, Auerbach R, see Globerson A kin AL, Steinhardt RA McGuigan JAS 27, 65 (212) 133,176 51, 52, 55, 64 Basten A, Miiller JFAP, Sprent Auld R, Alexander E, Levinsky Baker PF, McNaughton PA J, Cheers C (44) 170 N 93,103 52, 53, 64 Basten A, see Feldman M Avioli LV, see Bricker NS BakerPM (35) 117,121,169 (186) 123,124,175 96, 104 Balerna M, see Lazdunski M BatchelorJR (45) 151,152, Axe[rod AE, Prezansky J (23) 3, 69 170 165, 167,169 Baltimore D, see Goldstein G Batchelor JR, Chapman BA Axe[rod J, see Thoa NB 26, 73 (232) 141,177 (46) 151,170 Aynedjian HS, see Bank N Bank N, Koch KM, Aynedjian Bates RW, Laanes T, Riddle O 85, 86, 103 HS, Aras M 85, 86,103 (47) 163,170 Azar NA, Williams K, Takat- Banks RO, Kleinman LI Bathmann A, Land W, Koczo- suki K (24) 117,169 100, 103 rek K, Strass P (48) 146, Azer M, see Kaloyanides GJ Banks RO, see Kleinman LI 147,170 96,107 100,108 Batisto JR, see Goldstein AL Bannerjee S, see Goldstein AL (219) 137,176 Bach FH, see Sopori ML (474) (220) 136,176 Bauman EJ, see Marine D 124, 186 Barger AC 81,103 (344) 161,181 Bach JF (25, 26) 142, 143, Barger AC, Herd JA 77,103 Baumber JS, see Schneider EG 169 Barger AC, Yates FE, Rudolph 101,111 Bach JF, Carnaud C (27) AM 76,103 Baxter JD, Harris AW (49) 142, 169 Barhanin J, see Lazdunski M 157, 170 Bach JF, Dardenne M (28) 3, 69 Bayard F, see Beitens IZ 142, 169 Bariety J, see Papanicolao u N 100, 103 Bach JF, Dardenne M, Davies 97,110 Bealmar M, see Wilson R (543) AJS (29) 117,169 Barnes JL, see Reineck HJ 117,189 Bach JF, Dardenne M, Gold- 92, 111 Beaug6 L, see Di Polo R stein AL, Guha A, White A Barnola FV, see Villegas J 52, 53, 66 (30) 137, 142, 169 12,74 Beauvieux Y (50) 125,170 Bach JF, Dardenne M, Pleau BaxoniC (36) 154, 163,169 Bedarda G, see Buscarini L JM, Bach MA (32) 142, I69 Baroni C, Fabris N, Bertoli G (81) 135,171 Bach JF, Dural D, Dardenne (37) 154,169 Beeler GW, Reuter H 15, 31, M, Salomon JC, Tursz T (33) Baxoni C, Pesardo PC, Bertoli 51, 61, 65 142, 143,149,169 G (38) 154,169 Beitens IZ, Bayard F. Levitsky Bach JF, Papiernik M, Levas- Baroni C, see Pierpaoli W L, Ances IG, Kowanski A, seurP (31) 169 (413) 154, 184 Migeon CJ 100,103 Author Index 195

Bekkum DW van (51) 1 70 Berliner RW, see Brenner BM Blaese M, see Martinez C (347) Bekkum DW van, see Blank- 82, 90,104 117, 181 water MJ (63) 137,170 Berliner RW, see Dirks JH Blair NP, see Higgins JT Jr Bellanti JA, see Thong YA 90, 106 84, 85,107 (498) 157,187 Berliner RW, see Knox FG Blair RW, Peterson DF, Bishop Bello-Reuss E, Pastoriza-Munoz 84, 96, 98,108 VS 4, 65 R, Colindres RE 98, 103 Berliner RW, see Morgan T Blankwater MJ, Levert LA, Bello-Reuss E, Trevino DL, 90, 91,110 Swart AC, Bekkum DW van Gottschalk CW 99,103 Bernard C, Gargouil YM (63) 137,170 Belsky JL, see Papper S 36, 65 Blankwater MJ, see Krisbeek 85,110 Bernard P, Couraud F AM (307) 134, 180 Benforado JM 3, 4, 19, 65 12, 45, 46, 65 Blantz RC, Katz MA, Rector Bengele HH, Houttuin E, Bertoli G, see Baxoni C FC Jr, Seldin DW 78, 104 Pearce JW 96, 98, 104 (37, 38) 157, 169 Blantz RC, see Tucker BJ Bengele HH, Solomon S Besedowsky H, Sorkin E 87, 112 99, 104 (54) 147, 160, 164, 167, Blantz RC, see Wallin JD Bengele HH, see Misanko BS 170 76, 78,112 100, 110 Besedowsky HO, see Pierpaoli Blaufox MD, see Goldsmith DI Ben-Ishay D 89, 103 W (407, 408) 140, 160, 99, 106 Benios J, see Waltman SR 184 Blaustein MP 52, 65 (583) 152, 191 Best PM, see Baer M 31, 37, Blaustein MP, Hodgkin AL Bennacerraf B, see Katz DH 59, 64 52, 65 (282) 112, 179 Better OS, Massry SG 102, Blaustein MP, see Baker PF Bennett CM 91,104 104 51, 52, 55, 64 Bennett CM, Brenner BM, Bezvershenko I, Giulling E, Blecher M, White A (64) Berliner RW 82, 104 Dyugovskaya L (560) 157, 170 Bennett CD, see Scriabine A 135,190 Bleifer KH, see Papper S 49, 72 Bhattacharyya M, see Vassalle 85,110 Benninger C, Einw~ichter HM, M 22,74 Blinks JR, see Allen DG Haas HG, Kern R 55, 65 Bhattarai R, see Talwar GP 56,64 Bentzel C J, Anagnostopoulos (490) 162,187 Blomgren H, Anderson B T, Pandit H 84, 104 Bianchi CP, see Basseches P (65, 66) 126,149,170 Benzakein F, see Gutman Y 26, 64 Blomgren H, see Goldstein P 84, 85,107 Bianchi E, Pierpaoli W, Sorkin (233) 177 Beresewicz A, Reuter H 60, 65 E (55) 160,170 Bloom BR (67) 117,171 B~ress L, B6ress R, Wunderer Bianchi E, see Pierpaoli W Blythe WB, D'Avila O, Gitel- G 6,65 (415) 118, 160,184 man HJ, Welt LG 96,104 B6ress L, Wunderer G, Wach- Bianchi E, see Sorkin E (475) Blythe WB, see Klemmer PJ terE 6,65 160, 163,186 88,108 B~ress L, see Alsen C 46, 64 Biglieri EG, Forsham PH B6nisch H, Graefe KH, Keller B~ress L, see Rathmayer W 95,104 B 26, 65 11, 71 Billingham RE, Brent L (57) Boer V, see Milcu S (357) B~ress R, see B~ress L 6, 65 117, 170 135,182 Berglund K (52) 146,170 Billingham RE, Krohn PL, Bohidar NR, see Scriabine A Berglund K, Fagreus A (53) Medawar PB (58) 147, 1 70 49, 72 146, 170 Billingham RE, see Wilson DB Bohnenkamp H 23, 65 Berglund K, see Fagreus A (542) 120,189 Bomskov 144 (180) 175 Binimbo-Massengo A, see Bona DR Di, Chen LC, Sharp Bergmann C, Dubois JM, Deschaux P (142) 165,173 GWG 84, 85,105 Rojas E, Rathmayer W Bioquet Y, see Pleau JM (417) Bona GF Di, see Kaloyanides 11, 65 142, 184 GJ 96, 97, 107 Berliner DC, see Dougherty Biozzi G, Stiffel C, Mouton D, Bono E de, see Munck O TF (150) 149, 174 Liacopoulos-Briot M (59) 170 77,110 Berliner DL, see Dougherty Birge SJ, see Bricker NS Bonvalet JP, see Chabardes D TF (151) 149, 174 96,104 81,105 Berliner ML, see Dougherty Bishop VS, see Blair RW 4, 65 Bonvalet JP, see Poujeol R TF (150) 149,174 Bisset KA (60) 147,170 81,111 Berliner MZ, see Dougherty Bjornboe M, Fischel EL, Bonvalet JP, see Rouffignac TF (151) 149,174 Stoerk HC (61) 146,170 C de 81,111 Berliner RW, see Bennett CM Bjornboe M, Gormsen H, Lund- Boonjaxen S, see Stein JH 82, 104 quist LF (62) 117, 155, 170 91,112 196 Author Index

Borman SC, see Kunau RT Jr Brenner BM, Daugharty JM, Bruns F J, Alexander EA, 86, 91,108 Ueki IF, Troy JL 82,104 Riley AL, Levinsky NG Bose TD Du Jr, see Higashi- Brenner BM, Falchuk KH, 78, 79, 81,104 hara E 97, 107 Keimowitz RI, Berliner RW Bruns FJ, see Migdal S 78,110 Bottoner GD, Stitsch RD, 82, 104 Buckalew VM, Puschett JB, Roesel OF (68) 157,171 Brenner BM, Troy JL 82, 104 Kintzel JE 85,104 Bottoner GD, see Shaskar JR Brenner BM, Troy JL, Daug- Buckalew VM Jr, Martinez F J, (466) 156,186 harty TM 80, 104 GreenWE 96,104 Boulet P, see Carriere S Brenner BM, see Bennett CM Buckalew VM Jr, Walker BR, 81,105 82,104 Puschett JB, Goldberg M Boulpaep EL 84,104 Brenner BM, see Daugharty 90,104 Boulpaep EL, see Maunsbach TM 79, 80, 83,105 Buckalew VM Jr, see Gruber AB 84,109 Brent L, see Billingham RE KA 96,107 Bovee KC, Webster GD (57) 117,170 Buntner B, Szymik M (79) 77,104 Bricaine H, see Strauch G 160,171 Boyett JD, Hoffert JF (69) (480) 164,186 Burg C, see Wioland M (545) 157, 171 Brick HC (56) 158,170 125,189 Boylan JW, see Gertz KH Bricker NS, Klahr S, Purker- Burg MB, Orloff J 90,105 80, 106 son M, Schultze RG, Avioli Burger M, see Trainin M Boyle W, see Wagner H (521) LV, Birge SJ 96,104 (507,508) 138,139,188 121,134,188 Bridges JB, see Camblin JG Burke TJ, Robinson RR, Boylston AW, see Carpenter (83) 135,171 Clapp Jr 84,105 CB (87) 135,171 Brinck-Kohnson T, Dougherty Burke TJ, see Marchand GR Boyse EA, see Cantor H (84, TF (76) 171 77, 109 85, 86) 121,126,127,171 Brinck-Johnson T, see Munk A Burleson R, Levey RH (80) Boyse EA, see Goldstein G (383) 156,157,183 133,171 (229) 177 Broberger O, see Aperia A Burnett JC Jr, see Knox FG Boyse EA, see Komuro K 99,103 93, 95,108 (295, 296) 138, 141,172 Brody TM, see Akera T Buscarini L, Bedarda G, Boyse EA, see Scheid MP 42, 43, 56, 64 Marandola P (81) 135,171 (450) 136,141,185 Brody TM, see Ku DD Bush BMH, see Lowe DA Boyse EA, see Schlesinger M 42, 43, 69 12, 69 (461) 141,186 Bronsky EA, see Claman H Bussand A, see Aschkenasy A Bozler E 18, 65 (100) 119, 172 (18) 146,169 Brandis H, see Sethi KK (465) Brooks CMcC, Hoffmann BF, Buu NT, see Faucheux B 123,186 Suckling EE, Orias O 19, 65 99, 106 Brandis M, see Gertz KH Brooks CMcC, see Matsuda K By AW, see Kupchan SM 80, 106 19, 69 4, 5, 69 Braun G, see Gertz KH Brosio JL, see Thurman GB Bykovskaya SM, see Sergeev 90, 106 (499) 126,187 AV (464) 165,186 BraunW (70) 124, 158,171 Brostoff J, see Roitt IM (436) Byrom NA, Campbell MA, BraunW, Cohen EP (71) 119,185 Staughton RCD (82) 138, 171 Brown GL, Rothlauf MW, 171 Braun W, Ishizuka M (72) Heimzelling RH, Myers TS 158,171 (77) 148,157,171 Calahan MD 11, 66 Braun W, Ishizuka M, Matsu- Brown PR, Koutsaimanis KG, Camblin JG, Bridges JB (83) moto T (74) 158,171 Wardener HE de 96,104 135,171 Braun W, Ishizuka M, Win- Brude RM, see Waltman SR Campbeel R, see Munro AJ church R, Webb D (75) 171 (583) 152, 191 (384) 124, 183 Braun W, Rega MJ (73) 158, Brugh PM de, see Miller JFAP Campbell MA, see Byrom NA 171 (3711 117,182 (82) 138,171 Braun W, see Ishizuka K (266) Brunette MG, see Carrier S Campbell WB, see Higashihara 158,178 81,105 E 97,107 Braun W, see Winchurch R Brunner FP, Rector FC Jr, Cantor H, Boyse EA (84, 85) (544) 189 Seldin DW 90,104 121,126,127,171 Braun-Schubert G, see Gertz Brunner KT, Manuel J, Rudolf Cantor H, Shen FW, Boyse EA KH 80, 106 M, ChapinB (78) 122,171 (86) 127, 1 71 Brechtelsbauer H, see Keck W Brunner KT, see Cerrottini JC Cantor H, see Goldstein G 84,108 (93, 94) 121,172 (232) 141,177 Brenner BM, Berliner RW Brunner WH, see Claman H Cantor H, see Raff MC (425) 82, 90, 104 (103) 172 184 Author Index 197

Carafoli E, Crompton M Chase JH, see Dougherty TF Cline O, see Greengard R 51, 66 (149) 174 (241) 121. 157, 177 Carey RM 101,105 Chatworthy HW, see Ellison Code CF, see Dews PB (143) Carey RM, Smith JR, Ortt EM EH (170) 155,174 146,173 101,105 ChebotarevF (97) 147, 148, Coelho JB 81,105 Carey RM, see Lennane RJ 172 Coffey RG, see Sunshine GH 101,109 Cheers C, see Basten A (44) (488) 141,187 Carnaud C, see Bach JF (27) 170 Cohen EP, see Braun W (71) 142, 169 Cheever EV, see Lewis WJ 171 Carpenter CB, Boylston AW, (321) 163, 164,180 Cohen GH, see Hooper JS MerrileJP (87) 135,171 Chen C, Gettes LS, Katzung (258) 136,178 Carpenter CB, see Philipps SM BG 61, 66 Cohen I, see Attwell D (405) 135,184 Chen LC, see Di Bona DR 15, 22, 64 Carpenter CR, see Rubin AS 84, 85,105 Cohen J J, Claman HM (106) (440) 123,185 Chicheportiche R, see Laz- 126, 146,149, 172 Carriere S, Boulet P, Mathieu dunski M 3, 69 Cohen J J, Fischbach M, Claman A, Brunette MG 81,105 Childers JW, see Hanson RC HM (107) 146,149,172 Carriere S, Lamumiere G, 101,107 Cohen JJ, Hoope~ JA, Gold- Daigneault A, Champlain J de Chiller JM, Habicht GS, Weigl steinAL (108) 138, 172 99,105 WO (98) 120,172 Cohen JR, see Waksal D (524) Carsner RL, Rennels EG (88) Chisari FV, Edington TS (99) 127, 134, 188 163, 171 120,158,172 Cohen JT, Stavy L, Feldman Castro JE (89) 146,151,171 Chiti E, see Inzerillo R (263) M (109) 148,172 Castro JE, Hamilton DNH 178 Cohen KW, see Sunshine GH (90) 146,151,171 Christen R, see Agosier M (5) (488) 141,187 CatterallWA 3, 7, 11, 13, 14, 147,168 Cohen L, see Kaloyanides GJ 25, 66 Cirksena WJ, see Dirks JH 96, 97, 107 Catterall WA, see Galper JB 90,106 Cohen P, see Gershon RK 14,67 Cirksena WJ, see Schrier RW (206) 121,176 Ceglowski WS, Hand TL, 89,111 Cohn JN, see Velasquez MT Friedman H (92) 171 Claman H, Bronsky EA (100) 77, 78,112 Ceglowski WS, Labadie GW 119,172 Cole LJ, see Tyan HL (515) (91) 144,171 Claman H, Chaperton EA, 118,188 Ceglowski WS, see Hand TL Triplett RF (102) 118,172 Colindres RE, see Bello-Reuss (247) 144, 178 Claman H, McDonald W (101) E 98,103 Cerrottini JC, Brunner KT 119,172 Colley DG, Malakian A, Waks- (93) 121,172 Claman H, Moorhead JW, manB (110) 172 Cerrottini JC, Nordin AA, BrurmerWH (103) 172 Comsa J (111,112, 113, 114) Brunner KT (94) 121,172 Claman HM, Talmadge DW 118, 137, 140, 144,159, Chabardes D, Poujeol P, Deiss (104) 119,172 160, 161, 167,172 S, Bonvalet JP, Rouffignac Claman HM, see Cohen JJ ComsaJ, Filip G (115) 117, C de 81,105 (106,107) 126,146,149, 140, 172 Chamberlain SG, Kerkut GA 172 ComsaJ, Hook R (116) 125, 13, 66 Clampham WF, see Wardener 172 Champlain J de, see Carriere S HE de 79, 87, 94, 95, 96, Comsa J, Leonhardt H (117, 99,105 97,113 118, 119,120) 147,148, Charad J, Drueke T, Pujade- Clapp Jr, see Burke TJ 84,105 151,152, 153,155,156, Lauraine C, Lacoar B, Funck, Clark DA (105) 122, 172 172,173 Brentano JL 85, 105 Clark WR, see Lonai P (326) Comsa J, Leonhardt H, Chaourat G (95) 124, 172 120,181 OzminskiK (125) 140, 141, Chaperton EA, see Claman H Clark WR, see Ned_red J (388) 155, 173 (102) 118, 172 121,183 Comsa J, Leonhardt H, Chapin B, see Brunner KT Clarkson EM, Talner LB, Schwarz JA (123) 117, 140, (78) 122,171 Wardener HE de 96,105 154,155,173 Chapman BA, see Batchelor Clausen G, Tyssebotn I Comsa J, Philipp EM (121) JR (46) 151,170 81,105 137, 140, 144, 173 Chapman RA 55, 66 Clanstrat B, see Deschaux P Comsa J, Schwarz JA, Neu H Charlemagne J, see Fache B (562) 160, 190 (124) 140,144,149, 155, (176) 120, 175 Cleeman L, Morad M 28, 66 161,162, 173 Chase J, White A, Dougherty Clement DL, Pelletier CL, Comsa J, Schweissfurth R TF (96) 146,172 Shepherd JT 99,105 (122) 173 198 Author Index

Comstock JP, see Gabrourel Daly J J, Roe J, Harrocks P Davis BB, see Howards SS GD (199) 157,176 101,105 90, 107 Cone RE, Johnson AG (126) Daly JW, see Baxtels-Bernal E Davis BB, see Knox FG 158,173 38, 64 84, 96, 98, 108 Cone RE, see Marchalomis JJ Daly JW, see Shotzherger GS Davis BB, see Leeber DA (343) 158,181 39,73 90,109 Conger JD, Bartoli E, Earley Daly J, see Tokuyama T 5, 73 Davis C, see Aisenberg AC (6) LE 83,105 Dameshek W, see Wong FM 168 Constantio JS, see Goldstein (548) 189 Davis JM, see Schnermann J AL (225) 137,177 Damongsak D, see Hand TL 94,111 Coppleson LW, see Mosier DE (247) 144,178 Davis JO, see Johnston CI (378) 183 Daniel-Lamaziere JM, see 96,107 Coraboeuf E, Deroubaix E, Savart M (446) 156,185 Davis JO, see Schneider EG Coulombe A 16, 22, 38, 66 Danilo P, see Rosen MR 101,111 Corahoeuf E, Deroubaix E, 18,72 Defendi V, Roosa RA (133) Tazieff-Depierre F 44, 45, Dardenne M (561) 143,190 117,173 66 Dardenne M, Papiernik M, Deguchi T, see Narahashi T Corrado AP, Antonio A, Diniz Bach JF, Stutman O (128) 11, 70 CR 44, 66 162,173 DeHaan RL, McDonald TF, Cortell S, see Fitzgibbons JP Dardenne M, Pleau JM, Man Sachs HG 36, 66 87,106 MK, BachJF (129) 142, Deiss S, see Chabardes D Cortney MA 95,105 173 81,105 Cortney MA, Mylle M, Lassiter Dardenne M, see Bach JF (28, Deitmer JW, Ellis D 55, 66 WF, Gottschalk CW 90, 91, 29, 30, 32, 33) 117, 137, Delmasso AP, Martinez C, 105 142,143,149,169 Good RA (134) 117,133, Corvazier P, see Asehkenasy A Dardenne M, see Pleau JM 173 (18) 146,169 (417) 142, 184 Delmasso AP, Martinez C, Coulornbe A, see Coraboeuf E Dardenne MA, see Lacombe M Sjodink, Good RA (135) 16, 22, 38, 66 (309) 143,180 173 Couraud F, Rochat H, Lissitzky Darrach M, see Newson S Delmasso AP, see Martinez C S 37, 46, 54, 55, 66 (389) 147,148,155,183 (347) 117, 181 Couraud F, see Bernard P Daugharty JM, see Brenner Delmasso C, see Good RA 12, 45, 46, 65 BM 82,104 (234) 121,177 Couraud F, see Fayet G 45, 67 Daugharty TM, Ueki IF, Nicho- Delor B, see Senon B (463) Cowen DM, see Gowans JL las DP, Brenner BM 79, 80, 135,186 (237) 120,177 83,105 De Los Santos C, see Klemmer Cranefield PF 17, 18, 66 Daugharty TM, see Brenner PJ 88, 108 Crawford MK, see Thompson BM 80, 82, 104 Dennert G (136) 121,173 JS (497) 149, 156,187 Davidman M, Alexander E, Dennert G, see Tucker DF Crompton M, see Carafoli E Lalone R, Levinsky N (514) 121,188 51, 66 79, 80, 94,105 Deroubaix E, see Coraboeuf E Cross AM, Leuchars E, Miller Davidsohn J, see Stern K 16, 22, 38, 44, 45, 66 JFA (127) 118,134,161, (477) 151,186 Deschaux P (137) 149,159, 173 Davies AJS, see Bach JF (29) 160,161, 162,163,173 Cross AM, see Miller JFAP 117,169 Deschaux P, Claustrat B, Fon- (367,369) 118,182 Davies AJS, see Doenhoff MJ tanges R (562) 160, 190 Cuche J-L, see Diaz-Buxo JA (145) 125,173 Deschaux P, Dodin A, Laval F, 89,105 Davies AJS, see Elliott EV Fontange R (141) 140,173 Cuche J-L, see Ott CE 83,110 (169) 125,174 Deschaux P, Flores JL, Fon- Currie GA, see Grant CK (239) DaviesJS (130) 133,173 tange R (140) 159,162,173 177 Davies JS, Leuchaxs E, Wallis Deschaux P, Fontange R (138) Czaczkes JW, see Viscoper JR V, KollerPC (131) 119,173 173 96, 112 Davies JS, Leuchars E, Wallis Deschaux P, Fontange R, Gold- V, Marchant R, Elliot VE stein AL (563) 138,190 Daggett WM, see Gilmore JP (132) 173 Deschaux P, Morel G. Binimbo- 98,106 Davies WE, see Tyan HL Massengo A, Fontange R Daigneault A, see Carriere S (515) 118,188 (142) 165,173 99, 105 D'Avila O, see Blythe WB Deschaux P, Portier A, Fon- Dalheim H, see Schnermann J 96,104 tangeR (139) 173 94, 111 Davis BB, Knox FG, Wright Dev B, Drescher C, Schner- Daly J, Witkop B 37, 66 FS, Howards SS 89,105 mann J 94, 105 Author Index 199

Dews PB, Code CF (143) Dresser TP, see Knox FG Earley LE, Martino JA, 146, 173 89,108 Friedler RM 85,106 Diamantstein T (144) 157, Dresser TP, see Schneider EG Earley LE, see Bartoli E 173 93,111 80, 103 Diaz-Buxo JA, Haas JA, Ott Drueke T, see Chanard J Earley LE, see Conger JD CE, Cuche J-L, Marchand 85,105 83,105 GR, Knox FG 89,105 Drukker A, see Goldsmith DI Earley LE, see Friedler RM Diener E, see Lee KC (317) 99,106 77,106 119, 180 Dubois JB, see Senon B Earley LE, see Humphreys MH Dietrich FM, see Dukor P (463) 135,186 84, 85,107 (153) 146, 174 Dubois JM, see Bergmann C Earley LE, see Martino JA Diezi J, Michoud P, Aceves J, 11, 65 76, 77, 82, 86,109 Giebisch G 92, 106 Duchateau GH, see Gregoire C Earley LE, see Schrier RW Diezi J, Nenninger M, Gie- (242) 135,177 84,111 bisch G 91,106 Duclos TW, Kim BS (152) Earley LE, see Strauss MB Diniz CR, see Corrado AP 121,174 95, 112 44, 66 Dukor P, Dietrich FM (153) Eartley H, Leblond PC (164) Diniz CR, see Langer SZ 146,174 164, 174 44, 69 Dukor P, see Gisler R (208, East J, Parott DMV (165) Dirks JH, Cirksena WJ, Berli- 211) 119,123,176 134, 174 net RW 90, 106 Dukor P, see Leuchars E (318) East J, see Parrott D (400) Dirks JH, Seely JF 91,106 133,180 125,183 Doak SMA, see Miller JFAP Dukor P, see Miller JFAP Ebner F, Reiter M 60, 66 (367) 118, 182 (369,370) 182 Edelman IS, see Feldman M Dodin A, see Deschaux P Dumont F (154) 127,174 (188) 175 (141) 140,173 Dumont F, see Sabolevic EB Edelmann CM Jr, see Gold- Doenhoff MJ, Davies AJS, (442) 125,185 smith DI 99,106 Leuchars E, Wallis T (145) Dunn TD, Trainin N, Levey Edington TS, see Chisari FV 125, 173 RH (155) 159,174 (99) 120, 158, 172 Doherty, Shearer, Rosenthal Duquesnoy RJ (156,157) Edman P, see Rochat H 6, 72 131 118, 154,174 Egdahl RH, see Mannick JA Dolgin AE, see Wexter BC Duquesnoy RJ, Ahrens RC (342) 124,181 (538) 1.89 (158) 154,174 Eijsvogel VO, see Astaldi G Donatsch P, Lowe DA, Duquesnoy RJ, Mariani T, (21) 143, 169 Richardson BP, Tylor P Good RA (159) 154,174 Einw~chter HM, see Benninger 12,66 Duquesnoy RJ, see Kemp R C 55, 65 Doria G, Agarassi G, Pietro (285) 157,179 Eisen HN, Mayer MM, Moore SDi (146) 123,174 Dusing R, Melder B, Kramer DH, Tart RR, Stoerk HC Dougherty TF (147) 126, HJ 97, 106 (166) 146,174 146, 149,174 Dusing R, Opitz W-D, Kramer Eisental A, Nachtigal D, Feld- Dougherty TF, Berliner DC, HJ 97, 106 manM (167) 122,174 Berliner ML (150) 149,174 DuttonRW (160) 121,122, Eisner D, see Attwell D Dougherty TF, Berliner MZ, 123,174 15, 22, 64 Schneebeli G, Berliner DL Dutton RW, Falcoff R, Hirst Eknoyan G, Suki WN, Rector (151) 149, 174 JA, Hoffmann M (161) 174 FC Jr, Seldin DW 90, 106 Dougherty TF, Chase JH, Dural D, see Bach JF (33) Eldinger D, Garrett TJ (564) WhiteA (149) 174 142, 143,149, 169 190 Dougherty TF, White A (148) Duve C De (162) 157, 174 Elliott EV, Sinclair MR (168) 174 Dvorak HF, see Jankovic BD 146,174 Dougherty TF, see Brinck- (269) 124,178 Elliott EV, Weft V, Davies AJS Johnson T (76) 171 Dvoskfn B, see Makman MH (169) 125, 174 Dougherty TF, see Chase J (337,338,340) 157,181 Elliott EV, see Davies JS (132) (96) 146,172 Dwoskin B, see Pena A (402) 173 Dougherty TF, see Goldstein 183 EUiott EV, see Sinclair MR AL (220) 136, 176 Dyugovskaya L, see Bezver- (469) 117,186 Dougherty TF, see Hayes SP shenko I (560) 135,190 Elliott LH (565) 122, 190 (250) 146,155,178 Elhs D, see Deitmer JW 55, 66 Doyle H, see Wara W (529) Eardley DD, Gershon RK Ellison EH, Martin PC, Williams 137, 188 (163) 122,174 RD, Chatworthy HW, Hamwi Drescher C, see Dev B Earley LE, Friedler RM G, Zollinger RM (170) 155, 94,105 76, 77, 78, 85, 97,106 174 200 Author Index

Ellison EH, see Allen HL (9) Fauci AS, Pratt KR, Whalen G Fischel EE (190) 146, 155, 155,168 (567) 148,190 175 EUner CN, see Matsuda K Faulk M, see Perri WL (403) Fischel EE, Vaughan JH, Pho- 19, 69 183 topoulos K (191) 146, 175 Elster P, see McDougal JL Fayet G, Couraud F, Miranda Fischel EL, see Bjornboe M (572) 122, 190 F, Lissitzky S 45, 67 (61) 146, 170 Epstein FH, see Hayslett JP Feigelson M, see Feigelson P Fischer H, see Lohmann- 91,107 (181) 157,175 Mathes ML (324) 121,135, Erb P, Feldmann M (171) Feigelson P, Feigelson M 181 124, 175 (181) 157, 175 Fischer K, see Alsen C 46, 64 Ernst M, see Wekerle H (586) Fein RL, see Schrier RW Fisher B, see Fisher ER (192) 133,191 89,111 120, 175 Esparza A, see Young RH Feinstein MB, Paimre M Fisher ER, Fisher B (192) (552) 120,189 31,67 120, 175 Espiner EA, Tucci JR, Jagger Feldman M (182,183,184, Fitzgerald EM, see Obika LFO PI, Lauler DP 95,106 185) 119,120,149,175 101,110 Esselman WJ (172) 124,175 Feldman M, Basten A (186) Fitzgibbons JP, Gennari J, Esselman WJ, Miller HC (566) 123,124,175 Garfinkel HB, Cortell S 124,190 Feldman M, Funder JW, Edel- 87,106 Esselman W J, see Miller HC manIS (188) 175 Flacke W, see Kupchan SM (358) 133; 138,164,182 Feldman M, Globerson A 4, 69 Eulitz M, see Wunderer G (187) 119,156,175 Fleckenstein A, see Kaufmann 6,74 Feldman M, Kliburn JG, Levy R 18,68 Evans MM, see Kalden JR J (189) 175 Florentin I, see Kiger M (289) (277) 154,179 Feldman M, see Cohen JR 135,179 Evans R, see Grant CK (240) (109) 148,172 Flores JL, see Deschaux P 121,177 Feldman M, see Eisental A (140) 159, 162, 173 (167) 122,174 Fontaliran F, see Papanicolaou Fabiato A, Fabiato F 58, 66 Feldman M, see Erb P (171) N 97,110 Fabiato F, see Fabiato A 124,175 Fontanges R, see Deschaux P 58, 66 Feldman M, see Galili R (201) (138-142, 562, 563) 138, Fabin M, see Pierpaoli W (413) 119,176 140, 159, 160,162, 165, 154, 184 Feldman M, see Kontiainen S 173, 190 Fabris H, see Pierpaoli W (414) (298) 180 Ford LF, Michclen HS (193) 184 Feldman M, see Lonai P (325, 125,175 Fabris N (173) 150, 175 326) 120,181 Forsharn PH, see Biglieri EG Fabris N, Piantanelli L, Feldman M, see Najarian JS 95,104 Muzzoli M (174) 154,175 (385,386) 120,123,183 Fosset M, Barry J de, Lenoir Fabris N, Pierpaoli W, Sorkin Feldman M, see Wagner H MC, LazdunskiM 14, 37, 53, E (175) 154, 156, 175 (521,522) 121,134,188 54, 55, 67 Fabris N, see Baroni C (37) Feldman M, see Waksal D Fosset M, see Lazdunski M 154,169 (524) 127,134,188 3, 69 Fabris N, see Sorkin E (475) Ferrier GR 18, 6 7 Fosset M, see Romey G 160, 163,186 Ferris T, see Stein JH 91,112 12, 13, 37, 47, 48, 55, 72 Facile B, Charlemagne J (176) Ferris TF, see Stein JH Fox CA, Whitehead RH (195) 120, 175 78, 80,112 146, 175 Fachet J, Pakovits M, Vallant Fert C, see Plescia O (418) Fox KE, Gahourel JD (194) K (178) 175 158,184 157,175 Fachet J, Stark E, Valiant K, Field A, see Martinez D (574) Fozzard HA, see Walton M Pakovits M (177) 159,175 142,143,190 15,74 Fagreus A (179) 146,148,175 Fieser LF, Fieser M 4, 67 Fraig G, see Kenny JF (287) Fagreus A, Berglund K (180) Fieser M, see Fieser LF 4, 67 151,179 175 Filip G, see Comsa J (115) Frank M, see Akera T Fagreus A, see Berglund K 117,140,172 42, 43, 64 (53) 146,170 Fill JR Jr, Mason DT, Bartter Frank M, see Ku DD 42, 43, 69 Falchuk KH, see Brenner BM FC 99,106 Frankenhaeuser B, Hodgkin 82, 104 Finland M, see Kass EH (280) AL 14, 67 Falcoff R, see Dutton RW 147, 179 Franzl RB, see McMaster PD (161) 174 Finsterer U, see Keck W 84,108 (335) 146, 181 Faucheux B, Buu NT, Kuchel Fischbach M, see Cohen JJ Frazinova JB, see Meshalova O 99,106 (107) 146,149,172 AM (352) 146, 182 Author Index 201

Frelin C, see Lazdunski M George HW, see Krayer O Globerson A, Auerbach R 3, 69 4, 69 (212) 133, 176 Friedler RM, Earley LE Germuth FG (202) 176 Globerson A, Kirov SM, Parish 77, 106 Germuth FG, Oyama J, Otten- CR (214) 127, 176 Friedler RM, see Earley LE get B (203) 146, 155,176 Globerson A, Umiel T, Fried- 76, 77, 78, 85, 97, 106 Gershon RK (204, 205) manH (213) 140,176 Friedman H (196, 197, 198) 121,122, 127, 176 Globerson A, see Auerbaeh R 117, 123,176 Gershon RK, Cohen P, Hencin (22) 117,139, 169 Friedman H, see Ceglowski WS R, Liebhaber SA (206) 121, Globerson A, see Feldman M (29) 171 176 (187) 119, 156, 175 Friedman H, see Globerson A Gershon RK, see Eardley DD Globerson A, see Rotter V (213) 140, 176 (163) 122,174 (438, 439) 139, 185 Friedman H, see Hand TL Gertz KH, Brandis M, Braun- Globerson A, see Trainin M (247) 144, 178 Schubert G, Boylan JW (509) 117, 139, 188 Friedman WF, see Hurley JK 80,106 Goble FC, Konopka EA (215) 99, 107 Gertz KH, Mangos JA, Braun 151,176 Fritz H, see Wunderer G 6, 74 G, Pagel HD 90,106 Goldberg M, see Agus ZS Fry CH, see Bassingthwaighte Gettes LS, Reuter H 18, 67 98, 103 JB 27, 65 Gettes LS, see Chert C 61, 66 Goldberg M, see Buckalew VM Fujiwara M, Muramatsu I, Giebisch G, Klose RM, Wind- Jr 90, 104 Hidaka H, Ikushima S, hager EE 91,106 Goldman Y, see Morad M Ashida K 12, 50, 61, 67 Giebisch G, see Diezi J 57, 70 Funck-Brentano JL, see 91, 92,106 Goldsmith DI, Drukker A, Chanard J 85,105 Giebisch G, see Khuri RN Blaufox MD, Edelmann CM Funder JW, see Feldman M 91,108 Jr, Spitzer A 99, 106 (188) 175 Giebisch G, see Landwehr DM Goldsmith RS, see Schneider Furth R van, see Thompson JS 91,109 EG 98,111 (496) 151,187 Giesen GV, see Rector FC Jr Goldstein AL (216, 217) 79, 90, 94,111 116, 136, 138, 140, 176 Gabourel GD, Comstock JP Gill JR, see Alexander RW Goldstein AL, Asanuma Y, (199) 157, 176 99,103 Batisto JR, Hardy MA, Gabourel JD, see Fox KE Gillette R, see Gillette S Quint J, White A (219) (194) 157, 175 (568) 152,190 137, 176 Gabrielson A, Good RA (200) Gillette S, Gillette R (568) Goldsteitl AL, Bannerjee S, 146, 176 152, 190 Schneebeli GL, Dougherty Gabuzda G J, see Shear L Gilmore JP, Daggett WM TF, White A (220) 136,176 101,112 98,106 Goldstein AL, Guha A, Hardy Gafni M, see Ishizuka K (266) Gilmore JP, see Potkay S MA (223) 177 158,178 101,110 Goldstein AL, Guha A, Howe Galicich J, see McDonald KM Gisler R (207) 148,149,176 ML, White A (221) 138, 98, 109 Gisler R, Dukor P (208) 177 Galili R, Feldman M (201) 119,176 Goldstein AL, Guha A, Zatz 119,176 Gisler R, Schenkel-Hulliger L MM, Hardy MA, White A Galper JB, CatterallWA 14, 67 (209, 210) 154, 176 (222) 136, 177 Garby L, see Agarwal K (4) Gisler R, Staber F, Rude E, Goldstein AL, Slater FD, White 157,168 DukorP (211) 123,176 A (224) 136,164,177 Garfinkel HB, see Fitzgibbons Gitelman H J, see Blythe WB Goldstein AL, Thureau GB, JP 87,106 96,104 Rossio JL, Constantio JS Gargouil YM, see Bernard C Giulling E, see Bezvershenko I (225) 137,177 36, 65 (560) 135,190 Goldstein AL, White A (218) Garrett T J, see Eldinger D Glaser L, see Munson R 12, 70 116, 136, 176 (564) 190 Gleason SD, see Hanson RC Goldstein AL, see Asanuma Y Gasic G, see Agosier M (5) 101,107 (17) 169 147, 168 Gleason SD, see Obika LFO Goldstein AL, see Bach JF Gasser J, see Knox FG 91,108 101,110 (30) 137, 142, 169 Gauer OH, see Kapteina FW Glick G, see Arbel ER Goldstein AL, see Cohen JJ 101,108 17, 21, 22, 49, 64 (108) 138,172 Genant HK, see Katz AI Gliedman ML, see Mullane JF Goldstein AL, see Deschaux P 96, 108 102,110 (563) 138, 190 Gennari J, see Fitzgibbons JP Glitsch HG, Reuter H, Scholz Goldstein AL, see Hooper JS 87, 106 H 53, 55, 67 (258) 136, 178 202 Author Index

Goldstein AL, see Klein JJ Good RA, see Touraine JL Gupta PD, see Talwar GP (292) 179 (502, 503) 138,154,187 (490) 162, 187 Goldstein AL, see Kriiger J Good RA, see Yunis L (553) Gutman Y, Benzakein F (306) 118, 137,180 134,189 84, 85,107 Goldstein AL, see Law LW Goodwin T J, see Lennane RJ (315) 117, 137,180 101,109 Haan RL De, see McDonald Goldstein AL, see Markham Gorczynski RM, Miller RG, TF 36, 37, 70 RB (345) 147,181 Phillips RA (235) 123,177 Haas HG, see Benninger C 55, 65 Goldstein AL, see Paznino MM Gormsen H, see Bjornboe M Haas JA, see Diaz-Buxo JA (401) 137,183 (62) 117,155,170 89,105 Goldstein AL, see Schafer LA Gottschalk CW, see Allison M Haas JA, see Marchand GR (447) 138,185 100, 103 77,109 Goldstein AL, see Thurman Gottschalk CW, see Bello- Haas JA, see Ott CE 83,110 GB (499,500) 126,187 Reuss E 99,103 Habicht GS, see Chiller JM Goldstein AL, see Touraine JL Gottschalk CW, see Cortney (98) 120, 172 (503) 138,187 MA 90, 91,105 Habicht GS, see Tener GSL Goldstein AL, see Wara W Gowans JL, McGregor DD (582) 191 (529) 137,188 (236) 117,163,177 Hiigg LB, see Loor F (328) Goldstein AL, see Zissblatt M Gowans JL, McGregor DD, 126, 181 (558) 137,190 Cowen DM (237) 120, 177 H~immerling W, see Pollak A Goldstein G (226,227,228) Gowans JL, see McGregor DD (419) 184 141,157,177 (332) 120,181 Hakim AA, Lifson N 85,107 Goldstein G, Boyse EA (229) Grabar P, see Aschkenasy A Haksar A (244) 177 177 (18) 146,169 Halteman RH, see Lipman MF Goldstein G, Hoffman WW Graefe KH, see B6nisch H (322) 156, 180 (230) 141,177 26, 65 Hamilton DNH, see Castro JE Goldstein G, Manganaro A Graft RJ, Lappe M, Snell GD (90) 146,151,171 (231) 141,177 (238) 146,151,177 Hammar JA (245) 141,177 Goldstein G, Silvestroni AE, Graham HT, Grasser HS 8, 67 Hammerling W, see Scheid MP Cantor H, Baltimore D (232) Grant CK, Currie GA, Alexan- (450) 136, 141,185 141, 177 derP (239) 177 Hamwi G, see Ellison EH Goldstein G, see Basch RS Grant CK, Evans R, Alexander (170) 155,174 (42, 43) 119, 141,170 P (240) 121,177 Hanan R, Oyama J (246) Goldstein G, see Komuro K Grant GA, see Miller JFAP 119,178 (296) 141,179 (368,371) 117,182 Hanaoka M, see Nakao S Goldstein G, see Scheid MP Grasser HS, see Graham HT (387) 126, 183 (450) 136,141,185 8,67 Hand TL, Ceglowski WS, Goldstein G, see Schlesinger M Greaves MF, see Roitt IM Damongsak D, Friedman H (45,461) 141,186 (436) 119,185 (247) 144, 178 Goldstein G, see Sunshine GH Green WE, see Buckalew VM Hand TL, see Ceglowski WS (488) 141,187 Jr 96,104 (92) 171 Goldstein P, Wigzell H, Blom- Greengard R, Cline O (241) Hanin M, see Tanaka M 6, 73 gren H, Svedmyr EAJ (233) 121,157,177 Hanjan SHS, see Talwar GP 177 Greet MA, see Tasuda M (491) (490) 162, 187 Gonick HC, Saldanha LF 164,187 Hansen HJ, see Primus FJ 96, 107 Greger RF, see Marchand GR (423) 137, 184 Good RA, Delmasso C, Marti- 77, I O9 Hanson RC, McLane-Vega LA, nez C, Archer P, Pierce JC, Gregoire C, Duchateau GH Childers JW, Gleason SD, Papermaster BW (234) (242) 135,177 Schneider EG 10t, 107 121,177 Grill G, see Schnermann J Harada N, see Hotta Y 43, 68 Good RA, see Delmasso AP 94,111 Hardy MA, Quint AL, Slate D, (134, 135) 117,133,173 Groenewoud M, see Astaldi G White A (248) 137, 178 Good RA, see Duquesnoy RJ (21) 143,169 Hardy MA, see Goldstein AL (159) 154, 174 Gross AM, see Leuchars E (219, 222, 223) 136, 137, Good RA, see Gabrielson A (318) 133,180 176, 177 (200) 146,176 Gruber KA, Whitaker JM, Hargis BJ, see Malkiel S (341) Good RA, see Martinez C Buckalew VM Jr 96,107 146,155,181 (347,348) 117,181 Guha A, see Bach JF (30) Harris AW, see Baxter JD (49) Good RA, see Stutman O 137,142, 169 • 157, 170 (483, 484, 485,486,487) Guha A, see Goldstein AL (221) Harris AW, see Wagner H 117,133,134, 137,140,187 222, 223) 136,138,177 (522) 188 Author Index 203

Harris RH, Yarger WE 92, 107 Hidaka H, see Fujiwara M Hooper JA, see Cohen JJ Harrocks P, see Daly JJ 12, 50, 61, 67 (108) 138, 172 101,105 Higashihara E, Stokes JB, Hooper JS, McCaniel MC, Hartmann KW (249) 178 Kokko JP, Campbell WB, Thurman GB, Cohen GH, HalveRs E de, see Pollak A Du Bose TD Jr 97,107 Sculof RS, Goldstein AL (419) 184 Higgins JT Jr 89, 95,107 (258) 136,178 Hashimoto K, Ochi R, Hashi- Higgins JT Jr, Blair NP HopkinWJ (259) 122, 178 moto K, Inui J, Miura Y 84, 85,107 Hopkins P, see Young RH 6, 49,67 Hildeman WH, see Wistar R (552) 120,189 Hashimoto K, see Hashimoto (547) 148,189 Horackova M, Vassort G K 6,49,67 Hille B 3, 14, 15, 67 19, 20, 23, 24, 55, 59, 68 Hashimoto Y, Ashida K 6, 67 Hilleman MR, see Martinez D Hornych A, see Papanicolaou Ha Ty, Wathsman BH (243) (574) 142, 143,190 N 97,110 120, 137,177 Hillgard HR, see Yunis L Hornych A, see Richet G Haynes RC, see Sand H (443) (553) 134,189 84,111 157, 185 Hirst JA, see Dutton RW Horster M, Thurau K 81,107 Hayes RC, see Sutherland EW (161) 174 Horster M, see Schnermann J (489) 157, 187 Hochman PS, Kovacz G (257) 94, 111 Hayes SP, Dougherty TF 146,178 Horvat J, see Jankovic BD (250) 146,155,178 Hodgkin AL, Huxley AF (270) 144,179 Hays EF (251) 178 3, 7, 20, 67 Hoshi T, see Matsuda K Hays EF, Albert PF (252) Hodgkin AL, see Baker PF 40, 41, 70 117, 134, 178 51, 52, 55, 64 Hotta Y, Takeya K, Kobayashi Haystett JP, Kashgarian M, Hodgkin AL, see Blaustein MP S, Harada N, Sakakibara J, Epstein FH 91,107 52, 65 Shirai N 43, 68 Hayter CJ, see Wardener HE de Hodgkin AL, see Franken- Houssay BA, Sordelli A (260) 79, 87, 94, 95, 96, 97,113 haeuser B 14, 67 150,178 Heimzeiling RH, see Brown H6rhammer L, see List PH Houttuin E, see Bengele HH GL (77) 148, 157,171 5, 69 96, 98, 104 Heisler EM, see Schafer LA Hoffert JF, see Boyett JD (69) Howards SS, Davis BB, Knox (447) 138, 185 157,171 FG 90, 107 Heistracher P, Pillat B Hoffmann BF, see Brooks Howards SS, see Davis BB 17, 18, 40, 41, 42, 67 CMcC 19, 65 89, 105 Hellig H, Waldek JF (253) Hoffman BF, see Matsuda K Howards SS, see Johnston CI 146,178 19, 69 96,107 Hencin R, see Gershon RK Hoffmann MK, Watson J Howards SS, see Knox FG (206) 121,176 (569) 124,190 84, 96,108 Henney CS (254) 158,178 Hoffmann M, see Dutton RW Howe ML, see Goldstein AL Hensen SA, see Thong YA (161) 174 (221) 138, 177 (498) 157, 187 Hoffmann MK, see Scheid MP Howe R, Shanks RG 62, 68 Heppner RL, see Wood EH (451) 138,159,185 Howse JD, see Nutbourne DM 34, 74 Hoffman WW, see Goldstein G 96,110 Herbst AL, see Yates FE (550) (230) 141,177 Hiinig T, see Wecker E (532) 160, 189 Hogan PM, Albuquerque EX 123,157,188 Herd JA, see Barger AC 17, 37, 38, 49, 67 Humber DP, Pinder M, Hethe- 77,103 Holm C, see Perlman P (404) rington CM (570) 152, 190 Hermle M, see Schnermann J 184 Humphrey JH (261) 117,178 94,111 Holmes EW, DiScala VA Humphreys MH, Earley LE Herrera-Acosta J, AndreuCci 98,107 84, 85,107 VE, Rector FC Jr, Seldin Holzgreve H, Schrier RW Hunt SW, Mason DW, Williams DW 80,107 83,107 AF (262) 126,178 Herzenberg LA, see Sato VL Hondeghem LM, Katzung BG Hunter IC, see Stimson WH (445) 134, 185 15, 67 (578) 152, 191 Hess MW (255) 116,178 Honerjiiger P 8, 9, 23, 24, 25, Hurley JK, Kirkpatrick SE, Hess MW, Stoner RD (256) 26, 27, 30, 33, 67 Pitlick PT, Friedman WF, 117,178 Honerjiiger P, Meissner A 42 Mendoza SA 99,107 Hesselj6 R, see Sehellein Y Honerj/igerP, ReiterM 18, 23, Huxley AF, see Hodgkin AL (452) 162, 185 24, 25, 26, 28, 29, 30, 31, 3, 7, 20, 67 Hetherington CM, see Humber 32, 34, 35, 38, 39, 59, 60, DP (570) 152, 190 61, 62, 67, 68 Iijima T, Pappano AJ 36, 68 Hicks C, see Werthamer S Hook R, see Comsa J (116) Ikushima S, see Fujiwara M (537) 157, 189 125, 172 12, 50, 61, 67 204 Author Index

Ihle JM, see Paznino MM Jankovic BD, see Arnason BG Karambasis T, see Mountoka- (401) 137,183 (14) 117,168 lakis T 97,110 Incefy GS, see Touraine JL Jankovic BD, see Waksman BH Karema E, see Seibel K 60, 72 (502, 503) 138,154,187 (525) 117, 188 Kashgarian M, see Haystett Inesi G, see Johnson PN Jay EWK, see Wiesner K 6, 74 JP 91,107 24, 68 Jeejeebhoy HF (271) 117,179 Kass EH, Kendrick ML, Fin- Inui J, see Hashimoto K Jennings J, Taylor G (272) land M (280) 147,179 6, 49, 67 157,179 Kass RS, Lederer WJ, Tsien Inzerillo R, Chiti E (263) 178 Jerne NK, Mordin AA (273) RW, Weingart R 18, 68 Irisawa H, see Noma A 117,132,179 Kass RS, Tsien RW, Weingart 16, 42, 71 Johnson AG, see Cone RE R 18,68 Irisawa H, see Shigeto N (126) 158, 173 Katsuki S, see Shibata S 16,73 Johnson JA, see Schneider EG 48, 49, 72 Iriye R, see Kumazawa Z 101,111 Kattwinkel J, Munck A (281) 6, 69 Johnson PN, Inesi G 24, 68 157, 179 Irvine W J, see Kalden JR Johnston CI, Davis JO Katz AI, Genant HK 96,108 (277) 154, 179 96,107 Katz AM, Repke D 51, 68 Isakovic K, Shmith SB, Waks- Johnston CI, Davis JO, Katz DH, Bennacerraf B (282) man BH (264) 120, 178 Howards SS, Wright FS 112,179 Isakovic K, see Jankovic BD 96,107 Katz DM, see Amerding D (270) 144, 179 Jones B, Jones TC (274) (11) 123,137,168 Ischizuka M, see Winchurch R 121,179 Katz M, see Matsuda K 19, 69 (544) 189 Jones B, Jones TC, Rofff IM Katz MA, see Blantz RC Isenberg G 27, 68 (275) 121,179 78, 104 Ishima Y 36, 68 Jones HEH, see Kappas A Katz MA, see Wallin JD IshizukaK (265) 158,178 (279) 152,179 76, 78, 112 Ishizuka K, Gafni M, Braun W Jones JM, Kind PD (276) Katzung BG, see Chen C (266) 158,178 124,179 61, 66 Ishizuka M, see Braun W Jones TC, see Jones B Katzung BG, see Hondeghem (72, 74, 75) 158, 171 (274,275) 121,179 LM 15, 67 Israel E, see Schlesinger M Jones VE, see McGillivray MW Kaufmann R, Fleckenstein A, (460) 186 (331) 181 Antoni H, WolfH 18, 68 Israelit AH, see Mandin H Joppich R, see Keck W Kayaalp SO, Albuquerque EX, 80, 109 84,108 Warnick JE 37, 68 Ito K, Karaki H, Urakawa N Kaye AM, see Trainin M (508) 59, 68 Kahn JB, Acheson GH 36, 68 138,188 Iwamura N, see Shimizu T Kahn T, see Stein RM 90, 112 Kayibanda B, Rosenfeld C 17, 48, 49, 73 Kakisawa H, Kozima T, Yanai (283) 124, 179 Iwasa J, see Akera T 42, 43, M, Nakanishi K 6, 68 Keck W, Joppich R, Restoff 64 Kalden JR, Evans MM, Irvine WD v, Finsterer U, Pruck- Izumi T, see Shibata S WJ (277) 154,179 sunand P, Brechtelsbauer H, 48, 49, 72, 73 Kallskog O, Wolgast M Kramer K 84,108 87,108 Keeler RF, see Ohta M 4, 71 Jacques Y, see Lazdunski M Kaloyanides G J, Azer M Keimowitz RI, see Brenner 3, 69 96,107 BM 82, 104 Jacques Y, see Pouyss6gur J Kaloyanides GJ, Cohen L, Keiser HR, see Alexander RW 12, 71 Di Bona GF 96, 97,107 99, 103 Jacques Y, see Romey G Kamyama S, see Matsuda K Kelder D, see Ross SB 26, 72 12, 13, 72 40, 41, 70 Kelemen H, Lasmoler F, Jaff6 HL, Marine D (267) Kamm DE, Levinsky NG Milland G (284) 143,179 145, 178 88, 89,108 Keller B, see B6nisch H Jagger PI, see Espiner EA Kang DH, see Lee CO 56, 69 26, 65 95,106 Kao W, see Wiesner K 6, 74 Kemp R, Duquesnoy RJ (285) Jamison RL, Lacy FB Kaplan HS, Nagareda CS (278) 157,179 81,107 179 Kemp T, Marx L (286) 179 Janeway CA (268) 117,122, Kappas A, Jones HEH, Roitt Kemple KL, see Tanz RD 178 JM (279) 152,179 41,73 Jankovic BD, Dvorak HF Kapteina FW, Moth W, Kendrick ML, see Kass EH (269) 124, 178 Schwartz-Porsche D, Gauer (280) 147,179 Jankovic BD, Isakovic K, OH 101,108 Kenny JF, Pangburn PC, Fraig Horvat J (270) 144,179 Karaki H, see Ito K 59, 68 G (287) 151,179 Authorlndex 205

Kepinov L, Metalrtikov S Kleinman LJ, see Steichen JJ Kondo T, see Moore PF (374) (288) 150,179 100,112 147, 182 Kerkut GA, see Chamberlain Klemmer PJ, De Los Santos C, Konopka EA, see Goble FC SG 13, 66 Blythe WB 88, 108 (215) 151,176 Kern R, see Benninger C 55, 65 Klose RM, see Giebiseh G Kontiainen S, Feldman M Kersey B, see Martinez C 91,106 (298) 180 (348) 117,181 Klose RM, see Landwehr DM Kook AI, Trainin N (299, Ketelsen UP, see Wekerle H 91,109 300) 158,180 (585,586) 133,191 Knight TF, Weinman EJ Kook AI, Yakir Y, Trainin N Khodorov BI 3, 11, 68 90,108 (301) 138, 180 Khodorov BI, Revenko SV Knox FG, Burnett JC Jr, Kook AJ, see Trainin M (512) 9, 68 Kohan DE, Spielman WS, 188 Khuri RN, Wiederholt M, Strand JC 93, 95,108 Kopeyan C, Martinez G, Strieder N, Giebisch G Knox FG, Davis BB, Berliner Rochat H 6, 69 91,108 RW 98,108 Kopeyan C, see Martinez G Kidvaz Z, see Talwar GP Knox FG, Gasser J 91,108 6, 69 (490) 162, 187 Knox FG, Howards SS, Wright Kopeyan C, see Miranda F Kiger M, Flotentin I, Math6 G FS, Davis BB, Berliner RW 6, 70 (289) 135, 179 84, 96,108 Kopeyan C, see Rochat H Kill F, see Rector FC Jr Knox FG, Schneider EG, Dres- 6,72 79, 90, 94, 111 ser TP, Lynch RE 89,108 Kopin IJ, see Moss J 26, 70 Kilburn JG, see Feldman M Knox FG, Schneider EG, Kopin IJ, see Thoa NB (189) 175 Willis LR, Strandhoy JW, 26, 73 Kim BS, see Duclos TW (152) Ott CE 83, 90,108 Koppenh6fer E, Schmidt H 121,174 Knox FG, see Davis BB 10, 69 Kimberg DV, Loeb JH (290) 89,105 Korth M 58, 69 157, 179 Knox FG see Diaz-Bixo JA Koutsaimanis KG, see Brown Kimhi Y, see Trainin M (510) 89,105 PR 96,104 139,188 Knox FG, see Howards SS Kovacz G, see Hochman PS Kind PD, see Jones JM (276) 90,107 (257) 146,178 124, 179 Knox FG, see Marchand GR Kover G, see Oliw E 97,110 King DW, see Sanders GC 77, 109 Kowanski A, see Beitens IZ (444) 185 Knox FG, see Ott CE 100, 103 Kinney MJ, Di Scala VA 83,110 Kozima T, see Kakisawa H 78,108 Knox FG, see Schneider EG 6, 68 Kinoshita Y (291) 149,179 80, 93, 98,111 Kphn L, see Wecker E (532) Kintzel JE, see Buchalew VM Knutson F, Lundin PM (293) 123,157,189 85,104 162,179 Kramer HJ, see Dusing R Kirov SM, see Globerson A Kohayashi S, see Hotta Y 97,106 (214) 127,176 43,68 Kramer K, see Keck W Kirkpatrick SE, see Hurley JK Koch KM, see Bank N 84,108 99, 107 85, 86,103 Krayer O 4, 64, 69 Kirschenbaum MA, Stein JH Koch-Weser J 23, 26, 68 Krayer O, Acheson GH 97, 108 Koczorek K, see Bathman A 3,4,23,69 Kirshman D, see Sealey JE (48) 146,147, 170 Krayer O, George HW 4, 69 96,112 Kohan DE, see Knox FG Krayer O, Meilman E 4, 69 Klahr S, see Bricker NS 93, 95,108 Krayer O, see Moe GK 19, 70 96,104 Kokko JP, see Higashihara E Kreifner D 16, 42, 69 Klein H, see Stumpe KO 97,107 Kremshead JT, see North JR 93, 112 Koller PC, see Davies JS (131) (392) 183 Klein J J, Goldstein AL, White 119,173 Kretchmer RR, Perez-Tamayo A (292) 179 Komeva FA (294) 164, 166, R (302) 120, 180 Kleinerman J, see Shear L 172 Kreth HW, Williamson A (303) 101,112 Komuro K, Boyse EA (295) 180 Kleinman LI 100, 108 138,172 Krinsbeek AM (304) 180 Kleinman LI, Banks RO Komuro K, Goldstein G. Krisbeek AM, Astaldi GCB, 100, 108 Boyse A (296) 141,179 Blankwater MJ, Zijlstra JJ, Kleinman LI, Reuter JH Komuro K, see Scheid MP Levent LA, Astaldi A (307) 99, 108 (451) 138,159,185 134,180 Kleinman LI, see Banks RO Konda S, Stockert E, Smith Krohn PL (305) 147, 155, 100, 103 RT (297) 126,127, 180 180 206 Authorlndex

Krohn PL, see Billingham RE Lane P, see Philipps SM (405) Lee JB, see Attallah AA (58) 147,170 135,184 97,103 Kruck F, see Stumpe KO Lang FC, see Marchand GR Lee JS 85,109 93, 112 77,109 Lee KC (316) 149,180 Krtiger J, Goldstein AL, Waks- Langer SZ, Adler-Graschinsky Lee KC, Langman RE, Paltken manBH (306) 118,137, E, Almeida AP, Diniz CR VH, Diener E (317) 119, 180 44, 69 180 Ku DD, Akera T, Frank M, Langman RE, see Lee KC Lee KS, see Lee CO 56, 69 Brody TM 42,.43, 69 (317) 119,180 Leeber DA, Murdaugh HV, Ku DD, see Akera T Lappe M, see Graft RJ (238) Davis BB 90, 109 42, 43, 64 146,151,177 Lefkowitz I, see Watson J Kuchel O, see Faucheux B Laragh JH, see Sealey JE (584) 124, 191 99,106 96,112 Lehmkuhl D, see Sperelakis Kumazawa Z, Iriye R 6, 69 Larsson C, see Oliw W 97,110 N 53, 73 Kunau RT Jr, Lameire NH Laskov R (311) 180 Leicht R, Meves H, WellhSrner 86, 108 Lasmoler F, see Kelemen H HH 13, 69 Kunau RT Jr, Webb JL, Bor- (284) 143,179 Lennane RJ, Carey RM, Good- man SC 86, 91,108 Lassiter WF, see Cortney MA win TJ, Peart WS 101,109 Kunau RT Jr, see Stein JH 90, 91,105 Lennane RJ, Peart WS, Carey 92,112 Lauler DP, see Espiner EA RM, Shaw J 101,109 Kupchan SM, By AW 4, 5, 69 95,106 Lennox ES, see Tucker DF Kupchan SM, Flacke W 4, 69 Lauler DP, see McDonald M (514) 121,188 Kupchan SM, Zimmerman JH, 96,109 Lenoir MC, see Fosset M Alfonso A 4, 69 Lauler DP, see McDonald KM 14, 37, 53, 54, 55, 67 Kuschinsky W, Wahl M, 98,109 Leonhardt H, see Comsa J Wunderlich P, Thurau K Lauler DP, see Schrier RW (117-120, 123, 125) 117, 83,108 84, 86,111, 112 140, 147, 148,151 154, Laval F, see Deschaux P (141) 155, 156, 172, 173 Laanes T, see Bates RW (47) 140,173 Leskovits S, see McGillivray 163,170 Law LW (312, 313) 134,180 MW (331) 181 Labadie GW, see Ceglowski Law LW, Goldstein AL, White Leuchars E, Gross AM, Dukor WS (19) 144, 171 A (315) 117,137,180 P (318) 133,180 Labat M, Moroz C (308) 180 Law LW, Tranin N, Levey RH Leuchars E, see Cross AM Lackner LH, see Barton LJ (314,571) 133, 136, 180 (127) 118, 134, 161,173 91,103 190 Leuchars E, see Davies JS Lacombe M, Pleau F, Dardenne Law LW, see Levey RH (320) (131,132) 119, 173 MA, BachJF (309) 143, 134,180 Leuchars E, see Doenhoff MJ 180 Lawson Y, see Munro AJ (145) 125, 173 Lacour B, see Chanard J 85,105 (384) 124,183 Leuchars E, see Miller JFAP Lacy FB, see Jamison RL Lazdunski M 64 (369) 182 81,107 Lazdunski M, Balerna M, Bar- Leusen I, see Lameire N Lalone R, see Davidman M hanin J, Chicheportiche R, 88,108 79, 80, 94, 105 Fosset M, Frelin C, Jacques Levasseur P, see Bach JF (31) Lalone RC, see Levinsky NG Y, Lombet A, Pouyss6gur J, 169 79,109 Renaud JF, Romey G, Levent LA, see Krisbeek AM Lalurniere G, see Carriere S Schweitz H, Vincent JP 3, 69 (307) 134,180 99,105 Lazdunski M, see Fosset M Levert LA, see Blankwater MJ Lameire N, Matthys E, Mussche 14, 37, 53, 54, 55, 67 (63) 137,170 M, Ringoir S, Leusen I Lazdunski M, see Martinez G Levey RH, Medawar PB (319) 88,108 6, 69 146,180 Lameire NH, see Kunau RT Jr Lazdunski M, see Pouss6gur J Levey RH, Trainin N, Law LW 86, 108 12,71 (320) 134,180 Lameire NH, see Osgood RW Lazdunski M, see Romey G Levey RH, see Burleson R 88, 110 11, 12, 13, 37, 47, 48, 55, (80) 133,171 Lampen M, see Pollak A (419) 72 Levey RH, see Dunn TD (155) 184 Lazzari J, see Arbel ER 159,174 Lance EM, Medawar PB (310) 17, 21, 22, 49, 64 Levey RH, see Law LW (314, 180 Leblond PC, see Eartly H 571) 133,136, 180, 190 Land W, see Bathmann A (48) (164) 164,174 Levine DZ, see Schnermann J 146, 147,170 Lederer WJ, see Kass RS 18, 68 94, 111 Landwehr DM, Klose RM, Lee CO, Kang DH, Sokol JH, Levinsky NG, Lalone RC Giebisch G 91,109 Lee KS 56, 69 79, 109 Author Index 20 7

Levinsky N, see Auld R Lohmann-Mathes ML, Fischer Maisin HF (336) 181 93,103 H (324) 121,135,181 Makman MH, Dvoskin B, Levinsky NG, see Bruns FJ Lombet A, see Lazdunski M White A (337,338) 181 78, 79, 81,104 3, 69 Makman MH, Nakagawa S, Levinsky N, see Davidman M Lonai P, Clark WR, Feldman Dvoskin B, White A (340) 79, 80, 94,105 M (326) 120, 181 157, 181 Levinsky NG, see Kamm DE Lonai P, Feldman M (325) Makman MH, Nakagawa S, 88, 89, 108 181 White A (339) 157,181 Levinsky NG, see Migdal S Lonai P, Rotter V, Mogilnes Malakian A, see Colley DG 78,110 B, Trainin N (327) 121, (110) 172 Levinthal BG, see Lipman MF 139, 181 Malgren J, see Schellein Y (322) 156,180 Loot F, Amstutz H, H~igg LB, (452) 162, 185 Levitzky L, see Beitens IZ Mayor KS, Roelants GE Malkiel S, Hargis BJ (341) 100, 103 (328) 126,181 146, 155,181 Levitt MF, see Stein RM Lovenberg W, see Alexander Man MK, see Dardenne M 90,112 RW 99,103 (129) 142, 173 Levo Y, see Trainin M (511) Lovingood CG, see Allen HL Mandi B (573) 160, 190 139, 188 (9) 155,168 Mandin H, Israelit AH, Rector Levy J, see Feldman M (189) Low PA, Wu Ch, Narahashi T FC Jr, Seldin DW 80, 109 175 12, 69 Manganaro A, see Goldstein G Levy M 93,109 Lowe DA, Bush BMH, Ripley (231) 141,177 Lewy JE, Windhager EE SH 12, 69 Mangos JA, see Gertz KH 82, 109 Lowe DA, see Donatsch P 90, 106 Lewy JE, see Windhager EE 12, 66 Manley OT, see Marine D 82, 113 Lowitz HD, see Stumpe KO (344) 161,181 Lewis WJ, Cheever EV, Van- 85,112 Mannick JA, Egdahl RH derlaanWD (321) 163,164, Loyning EW 77,109 (342) 124,181 180 Lucas Z J, see Walter SM Manuel J, see Brunner KT Liacopoulos-Briot M, see (527) 124,188 (78) 122,171 Biozzi G (59) 170 Lucharskaya LM, see Sergeev Marandola P, see Buscarini L Lichardus B, Pearce JW AV (464) 165,186 (81) 135,171 96,106 LuckeyTD (329) 116,137, Marchalomis J J, Cone RE, Lichardus B, see Pearce JW 138,143,144,155,168,181 Rolley RT (343) 158, 181 98,110 Luckey TD, Venugopal B Marchand GR 87, 99, 94,109 Liebhaber SA, see Gershon (330) 138,143,181 Marchand GR, Burke T J, Haas RK (206) 121,176 Liicke-Huhle C, see Ruthen- JA, Romero JC, Knox FG Lifson N, see Hakum AA stroth-Baur W (441) 125, 77,109 85,107 185 Marchand GR, Ott CE, Lang Lily F, see Zissblatt M (558) Lundh H, see Redfern P FC, Greger RF, Knox FG 137, 190 13, 71 77, 109 Linker-Israeli M, see Trainin M Lundin PM, see Knutson F Marchand GR, see Diaz-Buxo (505,507) 117,139,188 (293) 162,179 JA 89,105 Lipham E, see Allison M Lundquist LF, see Bjornboe M Marchant T, see Davies JS 100, 103 (62) 117, 155,170 (132) 173 Lipman MF, Halteman RH, Luton JP, see Strauch G (480) Mariani T, see Duquesnoy RJ Levinthal BG, Percy S, 164,186 (159) 154,174 Thomson E (322) 156,180 Luttermann JW, see Schafer Marine D, Manley OT, Bau- Lipman MF, see Thompson LA (447) 138,185 man EJ (344) 161,181 EB (495) 157,187 Lynch RE, see Knox FG Marine D, see Jaff~ HL (267) Lissitzky S, see Couraud F 89,108 145, 178 37, 46, 54, 55, 66 Lynch RE, see Schneider EG Markham RB, White A, Lissitzky S, see Fayet G 80, 92, 111 Goldstein AL (345) 147, 45, 67 181 Lissitzky S, see Miranda F Machleidt W, see Wunderer G Marsh D J, see Quinn MD 6,70 6,74 87,111 Lissitzky S, see Roehat H Maddox DA, Price DC, Rector Marshall RA, see Schrier RW 6,72 FC Jr 84,109 86,111 List PH, H6rhammer L 5, 69 Mahrota ND, see Talwar GP Martin PC, see Ellison EH Litvac G (323) 121,181 (490) 162,187 (170) 155,174 Loeb JH, see Kimberg DV Maires RC, see Potvorski EF Martin WJ, Miller JFAP (346) (290) 157,179 (422) 125,184 134,181 208 Author Index

Martinez C, Delmasso AP, Maunsbach AB, Boulpaep EL McManus JP, Whitfield JF Blaese M, Good RA (347) 84, 109 (333, 334) 144, 158,162, 117,181 Maximov A (349) 141,181 181 Martinez C, Kersey B, Paper- Mayer MM, see Eisen HN McManus JP, see Rixon RH master W, Good RA (348) (166) 146,174 (431) 185 117,181 Mayopoulou-Symvoulidou McManus JP, see Whitfield JF Martinez C, see Delmasso AP MG, see Mountokalakis T (539) 150, 157,189 (134,135) 117,133,173 97,110 McMaster PD, Franzl RB Martinez C, see Good RA Mayor KS, see Loor F (328) (335) 146,181 (234) 121,177 126,181 McNaughton PA, see Baker PF Martinez C, see Stutman O Mayor-Withery KS, see Roe- 52, 53, 64 (485) 187 lants GE (435) 126,134, McNeil JS, see Schrier RW Martinez C, see Yunis L (553) 185 89,111 134,189 McAllister RE, Noble D, Tsien Medawar PB (350) 120,182 Martinez D, Field A, Schwann RW 15, 70 Medawar PB, Sparrow EM H, Tyrell AA, Hilleman MR McCaniel MC, see Hooper JS (351) 134,135,143,146, (574) 142, 143,190 (258) 136,178 152, 155,182 Martinez F J, see Buckalew VM McCarthy DA, see Swain HH Medawar PB, see Billingham Jr 96,104 21,43, 73 RE (58) 147,170 Martinez G, Kopeyan C, McDonald J, see Bahlmann JS Medawar PB, see Lance EM Schweitz H, Lazdunski M 96,103 (310) 180 6, 69 McDonald KM, Rosenthal A, Medawar PB, see Levey RH Martinez G, see Kopeyan C Schrier RW, Galicich J, (319) 146,180 6,69 Lauler DP 98,109 Meilman E, see Krayer O Martinez-Maldonado M, see McDonald KM, see Schrier 4, 69 Rector FC Jr 90,111 RW 84, 86,111, 112 Meissner A, see Honerjfiger P Martino JA, Earley LE McDonald M, Schrier RW, 42 76, 77, 82, 86,109 Lauler DP 96,109 Melder B, see Dusing R Martino JA, see Earley LE McDonald R, see Primus FJ 97,106 85, 106 (423) 137,184 Mellors T, see Perri WL (403) Maxtino L de, see Primus FJ McDonald S J, Wardener HE de 183 (423) 137,184 85,109 Melman A, Massry SG Marx L, see Kemp T (286) McDonald TF, Sachs HG 102,109 179 36, 70 Mendez, see Zipes 21 Mason DT, see Fill JR Jr McDonald TF, Sachs HG, Mendoza SA, see Hurley JK 99,106 DeHaan RL 36, 37, 70 99,107 Mason DW, see Hunt SW (262) McDonald TF, Trautwein W Merikas G, see Mountokalakis 126, 178 15, 70 T 97,110 Massry SG, see Better OS McDonald TF, see DeHaan RL Merlet-Benichou C, Rouffig- 102, 104 36, 66 nac C de 99,109 Massry SG, see Melman A McDonald TF, see Trautwein Merrile JP, see Carpenter CB 102, 109 W 51, 73 (87) 135,171 Math6 G, see Kiger M (289) McDonald W, see Claman H Meshalova AM, Frazinova JB 135,179 (101) 119,172 (352) 146,182 Mathieu A, see Carriere S McDougal JL, Shen FW, Elster Metalnikov S, see Kepinov L 81,105 P (572) 122,190 (288) 150,179 Matsuda K, Hoffman BF, McDowell EC, see Smith PE MetcalfD (353-355) 146,182 Ellner CN, Katz M, Brooks (471) 163,186 Meres H, see Leicht R 13, 69 CMcC 19, 69 McGillivray MW, Jones VE, Michclen HS, see Ford LF Matsuda K, Hoshi T, Kame- LeskovitsS (331) 181 (193) 125,175 yama S 40, 41, 70 McGregor DD, Gowans JL Michoud P, see Diezi J Matsumoto T, see Braun W (332) 120,181 92, 106 (74) 158,171 McGregor DD, see Gowans JL Migdal S, Alexander EA, Bruns Matsumura M, Takaori S (236,237) 117,120,163, FJ, Riley AL, Levinsky NG 39, 41, 70 177 78,110 Matsuo T, see Shibata S McGuigan JAS, see Bassingth- Migeon CJ, see Beitens IZ 48, 49, 72 waighte JB 27, 65 100,103 Matthys E, see Lameire N McLane-Vega LA, see Hanson Milcu S, Potop J (356) 135, 88,108 RC 101,107 144,182 Maul VA, see Zaalberg OBVD McLean MJ, Sperelakis N Milcu S, Potop J, Boer V, Oli- (554) 189 36, 70 niciN (357) 135,182 Author Index 209

Milcu S, see Potop J (421) Mogilnes B, see Lonai P (327) Mumludahl KE, see Zahnd GR 117,145,184 121,139,181 (555) 164,190 Milland G, see Kelemen H Money WL, see Perri WL (403) Munck A (382) 183 (284) 143,179 183 Munck A, Brink-Johnson T Miller and Good 128 Mongo KG, see Roulet MJ (383) 156,157,183 Miller HC, Esselmann WJ 57, 72 Munck A, see Kattwinkel J (358) 133, 138,164,182 Moore DH, see Eisen HN (281) 157,179 Miller HC, Schniege SK, Rule (166) 146,174 Munck A, see Morita Y (375) A (359) 138,182 Moore JW, see Narahashi T 157,182 Miller HC, see Esselman WJ 31, 70 Munck A, see Mosher KM (566) 124,190 Moore PF, Kondo T, Oliver RJ (376,377) 157,182, 183 Miller JFAP (360-362) 117, (374) 147,182 Munck A, see Wira C (546) 118,120,133,182 Moorhead JW, see Claman H 156,189 Miller JFAP, Brugh PM de, (103) 172 Munck O, Bono E de, Mills IH Grant GA (371) 117,182 Morad M, Goldman Y 57, 70 77,110 Miller JFAP, Doak SMA, Cross Morad M, Trautwein W 57, 70 Munro A, see Waldmann H MA (367) 118,182 Morad M, see Cleeman L (526) 123,188 Miller JFAP, Grant GA, Roe 28, 66 Munro AJ, Taussig MJ, Camp- FJC (368) 117,182 Morales-Aguilera A, Vaughan beel R, Williams H, Lawson Miller JFAP, Leuchars E, Williams EM 43, 70 Y (384) 124,183 Cross AM, Dukor P (369) Mordin AA, see Jerne NK Munson R, Westermark B, 182 (273) 117,179 Glaser L 12, 70 Miller JFAP, Mitchell GF Morel G, see Deschaux P (142) Muramatsu I, see Fujiwara M (363-365) 118,119,134, 165, 173 12, 50, 61, 67 182 Morgan G, see Scriabine A Murdaugh HV, see Leeber DA Miller JFAP, Osoba D (366) 49, 72 90, 109 182 Morgan T, Berliner RW Mussche M, see Lameire N Miller JFAP, Osoba D, Dukor 90, 91,110 88,108 P (370) 182 Morita Y, Munck A (375) Muzzoli M, see Fabris N (174) Miller JFAP, see Cross AM 157,182 154, 175 (127) 118,134,161,173 Moroz C, see Labat M (308) Myers TS, see Brown GL (77) Miller JFAP, see Martin WJ 180 148, 157,171 (346) 134,181 Morris AA, see Scriabine A Mylle M, see Cortney MA Miller RG, see Gorczynski RM 49, 72 90, 91,105 (235) 123,177 Morrison SL, see Tener GSL Milliez P, see Papanicolaou N (582) 191 Nachtigal D, see Eisental A 97,110 Mosher KM, Young DA, (167) 122, 174 Mills IH, see Mtlnck O 77,110 Munck A (376,377) 157, Nadeau A, see Zahnd GR Mills IH, see Wardener HE de 182, 183 (555) 164,190 79, 87, 94, 95, 96, 97,113 Mosier DE, Coppleson LW Nagareda CS, see Kaplan HS Miranda F, Kopeyan C, Rochat (378) 183 (278) 179 H, Rochat C, Lissitzky S Mosier DE, Pierce W (379) Nagy E (575) 154,190 6, 70 119,134,183 Najarian JS, Feldman M (385, Miranda F, see Fayet G 45, 67 Moss J, Thoa NB, Kopin IJ 386) 120, 123,183 Miranda F, see Rochat H 6, 72 26, 70 Nakagawa S, see Makman MH Miranda F, see Zlotkin E Moth W, see Kapteina FW (339, 340) 157,181 44, 74 101,108 Nakamura J, see Rotter V Misanko BS, Bengele HH, Mountain JM (380) 148,183 (438,439) 139,185 Solomon S 100,110 Mountokalakis T, Karambasis Nakanishi K, see Kakisawa H Mitchell GF, see Miller JFAP T, Mayopoulou-Symvo ulidou 6, 68 (363-365) 118,119,134, MG, Merikas G 97,110 Nakao S, Namba Y, Hanaoka 182 Mouton D, see Biozzi G (59) M (387) 126,183 Mitchison MA (372) 120, 182 170 Namba Y, see Nakao S (387) Mitchison MA, see Raft MC Mowbray JF (381) 135,183 126,183 (427) 125,184 Mozhayeva GN, Naumov AP, Narahashi T 3, 13, 31, 70 Miura Y, see Hashimoto K Negulyaev YA, Nosyreva Narahashi T, Anderson NC, 6, 49, 67 ED 10,41, 70 Moore JW 31, 70 Miyamoto H, Racker E 56, 70 Miiller JFAP, see Basten A Narahashi T, Seyama I 10, 70 Moe GK, Krayer O 19, 70 (44) 170 Narahashi T, Shapiro BI, Moeschlin S, Baguerra R, MuUane JF, Gliedman ML Deguchi T, Scuka M, Wang Baguerra J (373), 146,155, 103,110 CM 11, 70 182 Mullins LJ 35, 70 Narahashi T, see Low PA 12, 69 210 AuthorIndex

Narahashi T, see Ohta M Ochwadt B, see Stumpe KO Paimre M, see Feinstein MB 4, 71 85,112 31,67 Narahashi T, see Starkus JG Ohta M, Narahashi T, Keeler Pakovits M, see Fachet J 10,73 RF 4, 71 (177, 178) 159, 175 Nase S, see Raft MC (427) Ohta M, see Attwell D Paltken VH, see Lee KC (317) 125,184 15, 22, 64 119, 180 Naumov AP, see Mozhayeva Ojeda C, see Attwell D Pandian MR, Talwar GP (397) GN 10, 41, 70 15, 22, 64 154, 162,183 Nazanine J, see Robert A Olinici N, see Milcu S (357) Pandit H, see Bentzel CJ (432) 148,185 135,182 84,104 Nedred J, Touton M, Clark Oliver R J, see Moore PF Pane WE, see Stobo JD (479) WR (388) 121,183 (374) 147,182 143, 186 Negulyaev YA, see Mozhayeva Oliw E, Kover G, Larsson C, Pangburn PC, see Kenny JF GN 10,41, 70 Anggard E 97,110 (287) 151,179 Nenninger M, see Diezi J Opitz W-D, see Dusing R Pantelouris S (398) 118,183 91,106 97,106 Papanicolaou N, Safar M, Neu H, see Comsal J (124) Orias O, see Brooks CMcC Hornych A, Fontaliran F, 140, 144, 149,155,161, 19, 65 Weiss Y, Bariety J, Milliez P 162, 173 Orkand RK, see Tang CM 97,110 New W, Trautwein W 34, 51, 12,73 Papermaster BW, see Good RA 70 Orloff J, see Burg MB (234) 121,177 Newson S, Darrach M (389) 90,105 Papermaster W, see Martinez C 147, 148,155,183 Orosz CG (393) 124,183 (348) 117, 181 Nicholas DP, see Daugharty Ortt EM, see Carey RM Papiernik M, see Bach JF (31) TM 79, 80, 83,105 101,105 169 Niedergerke R 55, 70 Osgood RA, see Stein JH Papiernik M, see Daxdenne M Nielsen EL (390) 163,183 92,112 (128) 162,173 Nielsen S, see Wortis HH (549) Osgood RW, Lameire NH, Pappano AJ 36, 71 117, 133,189 Sorkin MI, Stein JH Pappano AJ, see Iijima T Nishimoto AY, see Philipson 88,110 36, 68 KD 56, 71 Osgood RW, Reineck HJ, Pappano A J, see Sperelakis N Nissen OI 79, 110 Stein JH 81, 87, 88, 92, 110 12, 36,53,54, 73 Noble D, see McAlhster RE Osgood RW, see Reineck HJ Papper S, Belsky JL, Bleifer 15, 70 92,111 KH 85,110 Noma A, Yanagihara K, Osgood RW, see Stein JH Pardos P, see Strauch G (480) Irisawa H 16, 42, 71 78, 80, 91,112 164, 186 Nomttra M, see Abe T (1) Osoba D (394,395) 152, Parish CR, see Globerson A 147,148,168 153,183 (214) 127,176 Norden SK, Young DA (391) Osoba D, see Miller JFAP Parma R, see Reineck HJ 157,183 (366, 370) 182 92,111 Nordin AA, see Cerrottini JC Ott CE, Haas JA, Cuche J-L, Parott DMV, see East J (165) (94) 121,172 Knox FG 83,110 134, 174 North JR, Kremshead JT, Ott CE, see Diaz-Buxo JA Parrillo E (576) 146,190 Askonas BA (392) 183 89,105 Parrott D, Sousa M de (399) Norton TR, see Shibata S Ott CE, see Knox FG 125, 183 48, 49, 72 83, 90,108 Parrott D, Sousa M de, East J Norton TR, see Tanaka M Ott CE, see Marchand GR (400) 125, 183 6,73 77,109 Passo SS, Thornborough JR, Nosyreva ED, see Mozhayeva Ottenger B, see Germuth FG Rothboller AB 101,110 GN 10, 41, 70 (203) 146,155,1 76 Pastoriza-Munoz R, see Bello- Notargiacomo MV, see Velas- Owen JJ, see Wortis HH (549) ReussE 98,103 quez MT 77, 78,112 117, 133,189 Paulsen F, see 8chellein Y Nutbourne DM, Howse JD, Owen JT, Raft MC (396) 183 (452) 162, 185 Schrier RW, Talner LB, Oyama J, see Germuth FG Paznino MM, Ihle JM, Gold- Ventom MG, Verroust PJ, (203) 146,155,176 stein AL (401) 137,183 Wardener HE de 96,110 Oyama J, see Hanan R (246) Pearce JW, Lichardus B 119,178 98,110 Obika LFO, Fitzgerald EM, Ozminski K, see Comsa J Pearee JW, Sonnenberg H Gleason SD, Zueker A, (125) 140,141,155,173 98,110 Schneider EG 101,110 Pearce JW, Sonnenberg H, Ochi R, see Hashimoto K Pagel HD, see Gertz KH Veress AT, Ackerman U 6, 49, 67 90,106 96,110 Author Index 21 1

Pearce JW, see Bengele HH Pierpaoli W, see Sorkin E Prezansky J, see Axelrod AE 96, 98,104 (475) 160, 163,186 (23) 165, 167, 169 Peaxce JW, see Lichardus B Pietro S Di, see Doria G (146) Price DC, see Maddox DA 96,106 123,174 84,109 Pearce JW, see Veress AT Pilanke LM, see A1-Adra AR Primus F J, Martino L de, 95,112 (559) 122, 190 McDonald R, Hansen HJ Peart WS, see Lennane RJ Pillat B, see Heistracher P (423) 137,184 101,109 17, 18,40,41,42,67 Proppe D, see Alsen C 46, 64 Pelletier CL, see Clement DL Pinder M, see Humber DP Prucksunand P, see Keck W 99,105 (570) 152,190 84, 108 Pena A, Dwoskin B, White A Pitkins DH (416) 148,184 Pujade-Lauraine C, see Cha- (402) 183 Pitlick PT, see Hurley JK nard J 85,105 Peper K, Trautwein W 99,107 Purkerson M, see Bricker NS 40, 41, 71 Pitts B JR 56, 71 96,104 Percy S, see Lipman MF (322) Playfair JHL, see Roitt IM Puschett JB, see Agus ZS 156, 180 (436) 119,185 98,103 Perez-Tamayo R, see Kxetsch- Pleau F, see Lacombe M (309) Puscbett JB, see Buckalew mer RR (302) 120, 180 143, 180 VM 85,104 Perlman P, Holm C (404) 184 Pleau JM, Dardenne M, Bioquet Puschett JB, see Buckalew Perri WL, Faulk M, Shapiro E, Y, Bach JF (417) 142, 184 VM Jr 90, 104 Mellors T, Money WL (403) Pleau JM, see Bach JF (32) 183 142, 169 Quinn MD, Marsh DJ 87,111 Pesardo PC, see Baroni C (38) Pleau JM, see Dardenne M Quint AL, see Hardy MA 154, 169 (129) 142,173 (248) 137,178 Peterson DF, see Blair RW Plescia O, Yamamoto I, Shima- Quint J, see Goldstein AL 4, 65 wara T, Fert C (418) 158, (219) 137,176 Pfitzenmaler K, see Rollinghoff 184 MA (437) 122,185 Pollak A, HLrnmerling W, Racker E, see Miyamoto H Philipp EM, see Comsa J (121) Lampen M, Harveirs E de 56, 70 137, 140, 144,173 (419) 184 RaffMC (424) 125,184 Philipps SM, Carpenter CB, Polo R Di 51, 52, 66 Raft MC, Cantor H (425) Lane P (405) 135,184 Polo R Di, Beaug~ L 52, 53, 66 184 Philipson KD, Nishimoto AY Pollen DW, Scott AC, Wallace Raft MC, Nase S, Mitchison 56, 71 WFM 62, 71 MA (427) 125,184 Phillips RA, see Gorczynski Ponce Zumino A, see Ruiz- Raft MC, Wortis HH (426) RM (235) 123,177 CerettiE 16, 72 125, 184 Photopoulos K, see Fischel EE Pora E, Toma V, Wittenberger Raft MC, see Owen JT (396) (191) 146,175 C, Rusdea D (420) 141, 183 Piantanelli L, see Fabris N 184 Rathmayer W, B~ress L 11, 71 (174) 154,175 Pottier A, see Deschaux P Rathmayer W, see Bergmann C Pierce JC, see Archer OK (12) (139) 173 11, 65 117, 168 Portius HJ, Repke K 36, 71 Ravens U 46, 47, 51, 59, 61, Pierce JC, see Good RA (234) Potkay S, Gilmore JP 101,110 71 121,177 Potop J, Milcu S (421) 117, Reckard CR (428) 120,184 Pierce W, see Mosier DE (379) 145,184 Rector FC Jr, Giesen GV, Kill F, 119,134,183 Potop J, see Milcu S (356, Seldin DW 79, 90, 94,111 PierpaoliW (406) 119,184 357) 135,144, 182 Rector FC Jr, Sellman JC, Pierpaoli W, Baroni C, Fabin Potvorski EF, Makes RC Martinez-Maldonado M, M, Sorkin E (413) 154,184 (422) 125,184 Seldin DW 90, 111 Pierpaoli W, Besedowsky HO Poujeol R, Bonvalet JP, Rouf- Rector FC Jr, see Barratt LJ (407, 408) 140,160, 184 fignac C de 81,111 79, 80,103 Pierpaoli W, Bianchi E, Sorkin Poujeol P, see Chabardes D Rector FC Jr, see Barton LJ E (415) 118,160,184 81,105 91,103 Pierpaoli W, Fabris H, Sorkin Pouyss6gur J, Jacques Y, Laz- Rector FC Jr, see Blantz RC E (414) 184 dunski M 12, 71 78,104 Pierpaoli W, Sorkin E (409- Pouyss6gur J, see Lazdunski M Rector FC Jr, see Brunner FP 412) 117, 160, 184 3, 69 90,104 Pierpaoli W, see Bianchi E Prahn RT, see Algire GH (8) Rector FC Jr, see Eknoyan G (55) 160,170 120,168 90, 106 Pierpaoli W, see Fabris N Pratt KR, see Fauci AS (567) Rector FC Jr, see Herrea- (175) 154, 156,175 148,190 Acosta J 80, 107 212 Author Index

Rector FC Jr, see Maddox DA Rho YM, see Touraine JL Rojas E, see Bergmann C 84, 109 (502) 154,187 11, 65 Rector FC Jr, see Mandin H Richardson BP, see Donatsch Rola-Pleszczinski M, see 80, 109 P 12,66 Thong YA (498) 157, 187 Rector FC Jr, see Wallin JD Richer G, Hornych A 84,111 Rolley RT, see Marchalomis 76, 78,112 Riddle O, see Bates RW (47) JJ (343) 158, 181 Redfern P, Lundh H, Thesleff 163, 170 Rollinghoff MA, Pfitzenmaier S 13,71 Riley AL, see Bruns FJ K,Wagner H (437) 122,185 Reeves JP, Sutko JL 56, 71 78, 79, 81,104 Romero JC, see Marchand GR Rega MJ, see Braun W (73) Riley AL, see Migdal S 77, 109 158, 171 78,110 Romey G, Abita JP, Schweitz Reichman ME (577) 152, 190 Ringoir S, see Lameire N H, Wunderer G, Lazdunski M Reif AE, Allen JMV van (429) 88,108 11,72 125, 185 Ripley SH, see Lowe DA Romey G, Jacques Y, Schweitz Reilly R, see Thompson JS 12,69 H, Fosset M, Lazdunski M (497) 149, 156,187 Ritchi JM 3, 71 12, 13, 72 Reinberg T, see Alsen C Ritter MA (430) 149,150, Romey G, Renaud JF, Fosset 46, 64 185 M, Lazdunski M 12, 37, 47, Reineck H J, Parma R, Barnes Rixon RH, Whitfield JF, 48, 55, 72 JL, Osgood RW 92,111 McManus JP (431) 185 Romey G, see Lazdunski M Reineck H J, see Osgood RW Rixon RH, see Whitfield JF 3, 69 81, 87, 88, 92, 110 (539) 150, 157,189 Roosa RA, see Defendi V Reiter M 5, 18, 23, 28, 29, 32, Robb CA, see Schneider EG (133) 117,173 56, 57, 58, 60, 64, 71 101,111 Rosen MR, Danilo P 18, 72 Reiter M, Stickel F J, Weber S Robbins JB, see Tanz RD Rosenberry TL, see Bartels- 61, 71 41,73 Bernal E 38, 64 Reiter M, see Ebner F 60, 66 Robert A, Nazanine J (432) Rosenfeld C, see Kayibanda B Reiter M, see Honerj~iger P 148,185 (283) 124,179 18, 23, 24, 25, 26, 28, 29, Roberts S, White A (433) Rosenstreich DL, see Wahl SJ 30, 31, 32, 34, 35, 38, 39, 145,185 (523) 157,188 59, 60, 61, 62, 67, 68 Robey G (434) 143,144, 185 Rosenthal A, see McDonald Reiter M, see Seibel K 60, 72 Robinson RR, see Burke TJ KM 98,109 Renaud JF, see Lazdunski M 84,105 Rosenthal J, Simone PG, 3, 69 Rochat C, see Miranda F 6, 70 Silbergleit A 97,111 Renaud JF, see Romey G Rochat C, see Rochat H 6, 72 Ross SB, Kelder D 26, 72 12, 37, 47, 48, 55, 72 Rochat H, Rochat C, Kopeyan Rossio JL, see Goldstein AL Renne T, see Senon B (463) C, Miranda F, Lissitzky S, (225) 137,177 135, 186 Edman P 6, 72 Rothboller AB, see Passo SS Rennels EG, see Carsner RL Rochat H, see Couraud F 101,110 (88) 163,171 37, 46, 54, 55, 66 Rothlauf MW, see Brown GL Repke D, see Katz AM 51, 68 Rochat H, see Kopeyan C (77) 148,157,171 Repke K, see Portius HJ 6,69 Rotter V, Globerson A, Naka- 36, 71 Rochat H, see Miranda F 6, 70 mura J, Trainin M (438,439) Restoff WD v, see Keck W Rochat H, see Zlotkin E 139, 185 84, 108 44, 74 Rotter V, see Lonai P (327) Reuter H 15, 71 Roe FJC, see Miller JFAP 121,139,181 Reuter H, Scholz H 15, 32, 71 (368) 117, 182 Rotter V, see Trainin M (511) Reuter H, Seitz N 52, 71 Roe J, see Daly JJ 101,105 139,188 Reuter H, see Baer M 31, 37, Roelants GE, Mayor-Withery Rouffignac C de, Bonvalet JP 59, 64 KS (435) 126,134,185 81,111 Reuter H, see Beeler GW Roelants GE, see Loor F (328) Rouffignac C de, see Chabardes 15, 31, 51, 61, 65 126,181 D 81,105 Reuter H, see Beresewicz A Roesel OF, see Bottoner GD Rouffignac C de, see Merlet- 60, 65 (68) 157,171 Benichou C 99,109 Reuter H, see Gettes LS Roiff IM, see Jones B (275) Rouffignac C de, see Poujeol 18, 67 121,179 R 81,111 Reuter H, see Glitsch HG Roitt IM, Torrigiani G, Greaves Roulet MJ, Mongo KG, Vassort 53, 55, 67 MF, Brostoff J, Playfair JHL G, Ventura-Clapier R 57, 72 Reuter JH, see Kleinman LI (436) 119, 185 Rubin AS, Carpenter CR 99,108 Roitt JM, see Kappas A (279) (440) 123,185 Revenko SV, see Khodorov 152,179 Rude E, see Gisler R (211) BI 9, 68 Rojas E, Rudy B 31, 72 123,176 Author Index 213

Rudolf M, see Brunner KT Scheid MP, Hoffman MK, Schnermann J, Wright FS, (78) 122, 171 KomuoK (451) 138,159, Davis JM, Stackelberg W v, Rudolph AM, see Barger AC 185 Grill G 94, 111 76,103 ScheUein Y, Hesselj5 R, Paul- Schnermann J, see Dev B Rudy B, see Rojas E 31, 72 sen F, Malgren J (452) 94,105 Ruiz-Ceretti E, Ponce Zumino 162, 185 Schniege SK, see Miller HC A 16,72 Schellekens PG, see Astaldi G (359) 138, 182 Rule A, see Miller HC (359) (21) 143,169 Scholz H 58, 61, 72 138, 182 Schenkel-Hulligex L, see Gisler Scholz H, Yazikof E de 61, 72 Rumrich G, Ullrich KJ R (209, 210) 154,176 Scholz H, see Glitsch HG 83,111 Schimpl A, Wecker E (453, 53, 55, 67 Rusdea D, see Pora E (420) 454,455) 123,186 Scholz H, see Reuter H 141,184 Schimpl A, see Wecker E 15, 32, 71 Ruthenstroth-Baur W, Liicke- (532) 123,157,188 Schrek B (462) 149,186 Huhle C (441) 125,185 Schlesinger M (456,457) Schrier RW, Earley LE 125,126,186 84,111 Sabolovic D, see Wioland M Schlesinger M, Goldstein G Schrier RW, Fein RL, McNeil (545) 125,189 (458) 186 JS, Cirksena WJ 89,111 Sabolevic EB, Dumont F Schlesinger M, Goldstein G, Schrier RW, McDonald KM, (442) 125, 185 Scheid M, Boyse EA (461) Marshall RA, Lauler DP Sachs HG, see De Haan RL 141,186 86,111 36, 66 Schlesinger M, Shlonai-Kor- Schrier RW, McDonald KM, Sachs HG, see McDonald TF zash Z, Israel E (460) 186 Wells RE, Lauler DP 36, 37, 70 Schlesinger M, Yaron T (459) 84,112 Safar M, see Papanicolaou N 186 Schrier RW, see Holzgreve H 97,110 Schmidmeier E, see Schner- 83,107 Sakakibara J, see Hotta Y mann J 94,111 Schrier RW, see McDonald KM 43, 68 Schmidt H, Schmitt 0 10, 72 98, 109 Saldanha LF, see Gonick HC Schmidt H, see Adam KR Schrier RW, see McDonald M 96,107 10, 64 96,109 Salomon JC, see Bach JF (33) Schmidt H, see KoppenhSfer Schrier RW, see Nutbourne 142, 143, 149,169 E 10,69 DM 96,110 Sand H, Haynes RC (443) Schmidt RF 17, 40, 41, 72 Schultze RG, see Bricker NS 157,185 Schmitt 0, see Schmidt H 96,104 Sanders GC, King DW (444) 10,72 Schwann H, see Martinez D 185 Schneebeli G, see Dougherty (574) 142, 143,190 Sano T, see Yamagishi S TF (151) 149,174 Schwartz N, see Viscoper JR 16, 74 Schneebeli GL, see Goldstein 96,112 Sato K 86,111 AL (220) 136,176 Schwartz-Porsche D, see Sato VL, Waksal SD, Herzen- Schneider EG, Davis JO, Robb Kapteina FW 101,108 berg LA (445) 134, 185 CA, Baumber JS, Johnson Schwarz JA, see Comsa J Sattler RW, see Alsen C 46, 64 JA, Wright FS 101,111 (123, 124) 117, 140, 144, Savart M, Daniel-Lamaziere Schneider EG, Dresser TP, 149, 154, 155,161,162,173 JM (446) 156,185 Lynch RE, Knox FG 98, 111 Schweissfurth R, see Comsa J Sayana R, see Tatwar GP Schneider EG, Goldsmith RS, (122) 173 (490) 162, 187 Arnaud CD, Knox FG Schweitz H, see Lazdunski M Scala VA Di, see Holmes EW . 98,111 3, 69 98, 107 Schneider EG, Lynch RE, Schweitz H, see Martinez G Scala VA, see Kinney MJ Willis LR, Knox FG 80,111 6, 69 78, 108 Schneider EG, see Hanson RC Schweitz H, see Romey G Schafer LA, Goldstein AL, 101,107 11, 12, 13, 72 Luttermann JW, Heisler EM Schneider EG, see Knox FG Scinader JW, see Wagner H (447) 138, 185 83, 89, 90,108 (521) 121, 134,188 SchaUer RT, Stevenson JK Schneider EG, see Obika LFO Scott AC, see Pollen DW (448) 133,185 101,110 62, 71 Schaumburg BP (449) Schnermann J 91,111 Scriabine A, Arman CG van, 156, 185 Schnermann J, Davis JM, Morgan G, Morris AA, Ben- Scheid M, see Schlesinger M Wunderlich P, Levine DZ, nett CD, Bohidar NR 49, 72 (461) 141,186 Horster M 94,111 Scuka M, see Narahashi T Scheid MP, Goldstein G, Ham- Schnermann J, Hermle M, 11, 70 merfing W, Boyse EA (450) Schmidmeier E, Dalheim H Sculof RS, see Hooper JS 136,141,185 94,111 (258) 136,178 214 Author Index

Sealey JE, Kirshman D, Laragh Shaw J, see Lennane RJ Smith RT, see Konda S (297) JH 96,112 101,109 126, 127, 180 Seely JF, see Dirks JH 91,106 Shear L, Kleinerman J, Smith SB, see Isakovic K Selbel K, Karema E, Takeya K, Gabuzda GJ 101,112 (264) 120, 178 Reiter M 60, 72 Shen FW, see Cantor H (86) SneUGD (472) 151, 163, 186 Seifen E 19, 72 127, 171 Snell GD, see Graft R.I (238) Seitz N, see Reuter H 52, 71 Shen FW, see McDougal JL 146, 151, 177 Sekerin CE, see Abraham AD (572) 122, 190 Sokol JH, see Lee CO 56, 69 (2) 156,157,168 Shepherd JT, see Clement DL Solle H, see Stumpe KO Seldin DW, see .Barratt LJ 99,105 93,112 79, 80,103 Sherman JD, see Wong FM Solomon GE (473) 148,186 Seldin DW, see Barton LJ (548) 189 Solomon S, see Bengele HH 91,103 Shibata S, Izumi T, Seriguchi 99,104 Seldin DW, see Blantz RC DG, Norton TR 48, 73 Solomon S, see Misanko BS 78, 104 Shibata S, Norton TR, Izumi 100, 110 Seldin DW, see Brunner FP T, Matsuo T, Katsuki S Solomon S, see Sonnenberg H 90, 104 48, 49, 72 83,112 Seldin DW, see Eknoyan G Shibata S, see Shimizu T Sonnenberg H 92, 112 90, 106 17, 48, 49, 73 Sonnenberg H, Solomon S Seldin DW, see Herrera-Acosta Shigenobu K, Sperelakis N 83, 112 J 80, 107 36, 73 Sonnenberg H, see Pearce JW Seldin DW, see Mandin H Shigenobu K, see Sperelakis N 96, 98,110 80, 109 36, 73 Sonnenberg H, see Wilson DR Setdin DW, see Rector FC Jr Shigeto N, Irisawa H 16, 73 92, 113 79, 90, 94, 111 Shimawara T, see Plescia O Sopori ML, Alter BJ, Bach FH, Seldin DW, see Wallin JD (418) 158, 184 Bach ML (474) 124,186 76, 78,112 Shimizu T, Iwamura N, Sordelli A, see Houssay BA Sellman JC, see Rector FC Jr Toyama J, Yamada K, (260) 150, 178 90, 111 Shibata S 17, 48, 49, 73 Sorkin E, Pierpaoli W, Fabris Senesky D, see Agus ZS Shirai N, see Hotta Y 43, 68 N, Bianchi F (475) 160, 98,103 Shlonai-Korzash Z, see Schle- 163, 186 Senon B, Renne T, Sermlar R, singer M (460) 186 Sorkin E, see Arrenbrecht S Delor B, Dubois JB, Thierry Shotzberger GS, Albuquerque (16) 155,162, 169 C (463) 135, 18_6 EX, Daly JW 39, 73 Sorkin E, see Besedowsky H Sergeev AV, Bykovskaya SM, Shuyser M (467) 156,186 (54) 147, 160, 164,167, Lucharskaya LM (464) Silbergleit A, see Rosenthal J 170 165, 186 97,111 Sorkin E, see Bianchi E (55) Seriguchi DG, see Shibata S Silvestroni AE, see Goldstein 160, 170 48, 73 G (232) 141,177 Sorkin E, see Fabris N (175) Sermlar R, see Senon B (463) Simone PG, see Rosenthal J 154, 156, 175 135,186 97,111 Sorkin E, see Pierpaoli W Sethi KK, Brandis H (465) Simonsen M (468) 117,186 (410-415) 117, 118, 154, 123,186 Sinclair MR, Elliott EV (469) 160,184 Sevcik C, see Villegas J 12, 74 117,186 Sorkin MI, see Osgood RW Sevensen C, see Thompson JS Sinclair MR, see Elliott EV 88,110 (497) 149, 156,187 (168) 146,174 Sousa de 128 Seyama I 42, 60, 72 SininskiA (470) 160, 186 Sousa M de, see Paxrott D Seyama I, see Narahashi T Sjodin K, see Delmasso AP (399, 400) 125,183 10, 70 (135) 173 Sparks JC, see Susic D Shafner JB, see Arnason BG Sj6din K, see Wilson R (543) 97,112 (15) 117,169 117,189 Sparrow E (476) 147,186 Shanes AM 8, 17, 72 Slate D, see Hardy MA (248) Sparrow EM, see Medawar PB Shanks RG, see Howe R 137,178 (351) 134, 135,143,146, 62, 68 Slater FD, see Goldstein AL 152, 155,182 Shapiro BI, see Narahashi T (224) 136,164,177 Sperelakis N, Lehmkuhl D 11, 70 Small M, see Trainin M (506, 53,73 Shapiro E, see Perri WL (403) 509,510) 117,138,139, Sperelakis N, Pappano AJ 183 188 12, 36, 53, 54, 73 Sharp GWG, see Di Bona DR Smith JR, see Carey RM Sperelakis N, Shigenobu K 84, 85,105 101,105 36, 73 Shaskar JR, Bottoner GD Smith PE, McDowell EC Sperelakis N, see McLean MJ (466) 156,186 (471) 163,186 36, 70 Author Index 215

Sperelakis N, see Shigenobu K Stoerk HC, see Eisen HN Svetmoldavski GY, see Zinzar 36, 73 (166) 146,174 SM (557) 117,190 Spielmau WS, see Knox FG Stokes JB, see Higashihara E Szymik M, see Buntner B (79) 93, 95,108 97,107 160, 171 Spitzer A, Windhager EE Stoner RD, see Hess MW (256) 82, 112 117,178 Takaori S, see Matsumura M Spitzer A, see Goldsmith DI St Pierre R, see Waksal D 39, 41, 70 99,106 (524) 127, 134,188 Takatsuki K, see Azar NA (24) Spitzer A, see Windhager EE St Pierre RL, see WiUis-Carr 117,169 82, 113 JJ (540) 165,167, 189 Takeya K, see Hotta Y 43, 68 Sprent J, see Basten A (44) Strand JC, see Knox FG Takeya K, see Seibel K 60, 72 170 93, 95,108 Talmadge DW, see Claman HM Staber F, see Gisler R (211) Strandhoy JW, Williamson HE (104) 119, 172 123, 176 101,112 Talner LB, see Clarkson EM Stackelberg W v, see Schner- Strandhoy JW, see Knox FG 96,105 mann J 94, 111 83, 90,108 Talner LB, see Nutbourne DM StLrnpfli R, see Adam KR Strass P, see Bathmann A (48) 96,110 10, 64 146,147,170 Talwar GP, Hanjan SHS, Kid- Stark E, see Fachet J (177) St~auch G, Pardos P, Luton vaz Z, Gupta PD, Mahrota 159, 175 JP, Bricaine H (480) 164, ND, Sayana R, Bhattarai R Starkus JG, Narahashi T 186 (490) 162, 187 10,73 Strauss MB, Earley LE 95,112 Talwar GP, see Pandian MR Staughton RCD, see Byrom Strichartz GR, see Tang CM (397) 154, 162, 183 NA (82) 138, 171 12,73 Tanaka M, Hanin M, Yasunobu Stavy L, see Cohen JT (109) Strieder N, see Khuri RN KT, Norton TR 6, 73 148, 172 91,108 Tang CM, Strichartz GR, Steichen JJ, Kleinman LI Stumpe KO, Lowitz HD, Orkand RK 12, 73 100, 112 Ochwadt B 85,112 Tanz RD 41, 73 Stein JH, Osgood RW, Boon- Stumpe KO, Solle H, Klein H, Tanz RD, Robbins JB, Kemple jaren S, Ferris T 91,112 Kruck F 93,112 KL, Allen PA 41, 73 Stein JH, Osgood RW, Ferris Stutman (481,482) 126,127, Tart M 31, 73 TF 78, 80, 112 187 Tarr RR, see Eisen HN (166) Stein JH, Osgood R.A, Kunau Stutman O (579, 580,581) 146,174 RT Jr 92, 112 127,140,191 Tasuda M, Greet MA (491) Stein JH, see Kirschenbaum Stutman O, Good RA (483, 164, 187 MA 97, 108 484) 117,137,187 Taub RH, see Wong FM (548) Stein JH, see Osgood RW Stutman O, Yunis EJ, Good 189 81, 87, 88, 92, 110 RA (486,487) 133, 134, Taussig MJ, see Munro AJ Stein RM, Abramson RG, 140,187 (384) 124,183 Kahn T, Levitt MF 90,112 Stutman O, Yunis E J, Martinez Taylor G, see Jennings J (272) Steinhardt RA, see Baker PF C, Good RA (485) 187 157, 179 51, 52, 55, 64 Stutman O, see Dardenne M Taylor RB (492, 493) 117, Stern K, Davidsohn J (477) (128) 162, 173 119, 187 151,186 Suckling EE, see Brooks CMcC Taylor RB, Allison AC (494) Stetson CA (478) 120,186 19, 65 187 Stevenson JK, see Sehaller RT Suki WN, see Eknoyan G Tazieff-Depierre F, see Cora- (448) 133, 185 90,106 boeufE 44, 45, 66 Stickel FJ, see Reiter M 61, 71 Sulakhe PV, St Louis PJ 56, 73 Tener GSL, Morrison SL, Stiffel C, see Biozzi G (59) Sunshine GH, Baseh RS, Cof- Habicht GS (582) 191 170 fey RG, Cohen KW, Gold- Thesleff S, see Redfern P Stimson WH, Hunter IC (578) stein G (488) 141,187 13, 71 152, 191 Susie D, Sparks JC 97,112 Thierry C, see Dubois JB Stitsch RD, see Bottoner GD Sutherland EW, Hayes RC (463) 135,186 (68) 157,171 (489) 157,187 Thoa NB, Wooten GF, Axel- St. Louis PJ, see Sulakhe PV Sutko JL, see Reeves JP rod J, Kopin IJ 26, 73 56, 73 56, 71 Thoa NB, see Moss J 26, 70 Stobo JD, Pane WE (479) Swain HH, McCarthy DA Thodenius K, see Aperia A 143, 186 21, 43, 73 99, 103 Stoekert E, see Konda S (297) Swart AC, see Blankwater MJ Thomas L, see Weismann G 126, 127,180 (63) 137,170 (534, 535) 157,189 Stoerk HC, see Bjornboe M Svedmyr EAJ, see Goldstein P Thompson EB, Lippman MF (61) 146,170 (233) 177 (495) 157, 187 216 Author Index

Thompson E, see Lippman MF Trainin M, see Rotter V (438, Ulbricht W 3, 7, 8, 14, 20, 22, (322) 156, 180 439) 139,185 23, 29, 64, 73, 74 Thompson JS, Crawford MK, Trainin N, see Dunn TD (155) Ullmann TD, see Viscoper JR Reilly R, Sevensen C (497) 159,174 96, 112 149, 156, 187 Trainin N, see Kook AI (299, Ullrich K J, see Rumrich G Thompson JS, Furth R van 300,301) 138,158,180 83, 111 (496) 151,187 Tranin N, see Law LW (314, Umiel T, Trainin N (516) Thong YA, Hensen SA, Vin- 571) 133,136,180, 190 139,188 cent MM, Rola-Pleszczinski Trainin N, see Levey RH Umiel T, see Globerson A M, Walsh JB, Bellanti JA (320) 134,180 (213) 140, 176 (498) 157, 187 Trainin N, see Lonai P (327) Umiel T, see Trainin M (512) Thronborough JR, see Passo 121,139,181 188 SS 101,110 Trainin N, see Umiel T (516) Urakawa N, see Ito K 59, 68 Thurau K, see Horster M 139,188 Urquhard J, see Yates FE 81,107 Trautwein W 3, 7, 15, 59, 73 (550) 160, 189 Thurau K, see Kuschinsky W Trautwein W, McDonald TF, 83,108 TripathiO 51, 73 Valiant K, see Fachet J (177, Thureau GB, see Goldstein AL Trautwein W, see McDonald 178) 159,175 (225) 137,177 TF 15, 70 Vanderlaan WD, see Lewis WJ Thurman GB, Barr PS, Gold- Trautwein W, see Morad M (321) 163, 164,180 steinAL (500) 187 57, 70 Vann DC (517) 123,188 Thurman GB, Brosio JL, Gold- Trautwein W, see New W VassaUe M, Bhattacharyya M stein AL (499) 126,187 34, 51, 70 22, 74 Thurman GB, see Hooper JS Trautwein W, see Peper K Vassort G, see Horarkova M (258) 136,178 40, 41, 71 19, 20, 23, 34, 55, 59, 68 Tokuyama T, Daly J, Witkop Trevino DL, see Bello-Reuss E Vassort G, see Roulet MJ B5,73 99,103 57, 72 Toma V, see Pora E (420) Tripathi O, see Trautwein W Vaughan JH, see Fischel EE 141,184 51, 73 (191) 146,175 Torgyan S (501) 150, 187 Triplett RF, see Claman H Vaughan Williams EM, see Torrigiani G, see Roitt IM (102) 118,172 Morales-Aguilera A 43, 70 (436) 119,185 Troy JL, see Brenner BM Vaux de St Cyr C, see Axnason Touraine F, see Tottraine JL 80, 82,104 BG (15) 117,169 (502) 154,187 Trygzynski EW, see Wexler BC Velasquez MT, Notargiacomo Touraine JL, Incefy GS, Gold- (538) 189 MV, Cohn JN 77, 78, 112 stein AL, Good RA (503) Tse Hy (513) 127,188 Ventom MG, see Bahlmann JS 138, 187 Tsien RW, see Kass RS 18, 68 96,103 Touraine JL, Incefy GS, Tou- Tsien RW, see McAllister RE Ventom MG, see Nutbourne raine F, Rho YM, Good RA 15, 70 DM 96,110 (502) 154, 187 Tucci JR, see Espiner EA Ventura-Clapier R, see Roulet Touton M, see Nedred J (388) 95,106 MJ 57, 72 121,183 Tucker BJ, Blantz RC 87,112 Venugopal B, see Luckey TD Toyama J, see Shimizu T Tucker DF, Dennert G, Lennox (330) 138, 143,181 17, 48, 49, 73 ES (514) 121. 188 Veress AT, Pearce JW 95,112 Trainin M (504) 116,188 Tursz T, see Bach JF (33) Veress AT, see Pearce JW Trainin M, Burger M, Kaye 142, 143,149,169 96,110 AM (508) 138, 188 Twisk MJ, see Zaalberg OBVD Verroust PJ, see Nutbourne Trainin M, Burger M, Linker- (554) 189 DM 96, 110 IsraeliM (507) 139,188 Tyan HL, Cole LJ, Davies WE Vessey SH (518) 148,188 Trainin M, Kook AJ, Albuia M, (515) 118,188 Villegas J, Sevcik C, Baxnola Umiel T (512) 188 Tylor P, see Donatsch P FV, Villegas R 12, 74 Trainin M, Levo Y, Rotter V 12, 66 Villegas R, see Villegas J (511) 139,188 Tyrell AA, see Martinez D 12,74 Trainin M, Linker-Israeli M (574) 142, 143,190 Vincent JP, see Lazdunski M (505) 117, 139, 188 Tyssebotn I, see Clausen G 3, 69 Trainin M, Small M (506) 81,105 Vincent MM, see Thong YA 138, 188 (498) 157,187 Trainin M, Small M, Globerson Ueki IF, see Brenner BM Viscoper JR, Czaczkes JW, A (509) 117, 139,188 82,104 Schwartz N, Ullmann TD Trainin M, Small M, Kimhi Y Ueki IF, see Daugharty TM 96,112 (510) 139,188 79, 80, 83,105 Visscher TL (519) 149, 188 Author Index 217

Wachter E, see B6ress L 6, 65 Wara W, Ammann AJ (528) Wekerle H (536) 127,132, Wachter E, see Wunderer G 138,189 189 6,74 Wara W, Goldstein AL, Doyle Wekerle H, Ketelsen UP (585) Wagner H (520) 121,188 H, Ammann AJ (529) 137, 133,191 Wagner H, Feldman M, Boyle 188 Wekerle H, Ketelsen UP, Ernst W, Scinader JW (521) Wardener HE de, Mills IH, M (586) 133, 191 121,134,188 Clapham WF, Hayter CJ Wekerle H, see Waksal D (524) Wagner H, Harris AW, Feld- 79, 87, 94, 95, 96, 97,113 127, 134,188 man M (522) 188 Wardener HE de, see Bahl- WellhiSrner HH. see Leicht R Wagner H, see Rollinghoff MA mann JS 96,103 13, 69 (437) 122, 185 Wardener HE de, see Brown Wells RE, see Schrier RW Wahl M, see Kuschinsky W PR 96,104 84,112 83, 108 Wardener HE de, see Clarkson Welt LG, see Blythe WB Wahl SJ, Altmann LC, Rosen- EM 96,105 96,104 streich DL (523) 157,188 Wardener HE de, see McDonald Wennersten C, see Arnason BG Waksal D, Cohen JR, Waksal SJ 85,109 (14) 117,168 HW, Wekerle H, St Pierre R, Wardener HE de, see Nut- Werthamer S, Hicks C, Amarat Feldman M (524) 127,134, bourne DM 96,110 L (537) 157,189 188 Warnick JE, see Albuquerque Westermark B, see Munson R Waksal HW, see Waksal D EX 13, 64 12, 70 (524) 127,134,188 Warnick JE, see Kayaalp SO Wexler BC, Dolgin AE, Tryg- Waksal SD, see Sato VL (445) 37, 68 zynskiEW (538) 189 134, 185 Wathsman BH, see Ha Ty Whalen G, see Fauci AS (567) Waksman B, see Colley DG (243) 120, 137,177 148,190 (110) 172 Watson J, Aarden LA, Lefko- Whitaker JM, see Gruber KA Waksman BH, Arnason BG, witzI (584) 124,191 96, 107 Jankovic BD (525) 117, Watson J, see Hoffmann MK White A, see Asanuma Y (17) 188 (569) 124,190 169 Waksman BH, see Arnason BG Watts HG (530) 158,188 White A, see Bach JF (30) (14) 117,168 Weaver JM, see Algire GH (8) 137, 142,169 Waksman BH, see Isakovic K 120,168 White A, see Blecher M (64) (264) 120, 178 Webb D, see Braun W (75) 171 157,170 Waksman BH, see Kriiger J Webb D, see Winchurch R White A, see Chase J (96) (306) 118, 137,180 (544) 189 146,172 Waldek JF, see Hellig H (253) Webb DR (531) 158,188 White A, see Dougherty TF 146,178 Webb JL, see Kunau RT Jr (148, 149) 174 Waldmann H, Munro A (526) 86, 91,108 White A, see Goldstein AL 123,188 Weber S, see Reiter M 61, 71 (218-224) 116,136, 137, Walker BR, see Buckalew VM Webster GD, see Bovee KC 138,164,176, 177 Jr 90,104 77,104 White A, see Hardy MA (248) Walker SM, Lucas ZJ (527) Wecker E, Schimpl A, Hiinig T, 137,178 124,188 KphnL (532) 123,157,188 White A, see Klein JJ (292) Wallace WFM, see Pollen DW Wecker E, see Schimpl A 179 62, 71 (453,454,455) 123,186 White A, see Law LW (315) Wallin JD, Blantz RC, Katz Weidmann S, see Wood EH 117, 137,180 MA, Andreucci VE, Rector 34, 74 White A, see Makman MH FC Jr, Seldin DW 76, 78, Weigl WO, see Chiller JM (98) (337-340) 157,181 112 120, 172 White A, see Markham RB Waltin JD, see Barratt LJ Weil V, see Elliott EV (169) (345) 147,181 79, 80,103 125,174 White A, see Pena A (402) 183 Wallis T, see Doenhoff MJ Weingart R, see Kass RS White A, see Roberts S (145) 125,173 18, 68 (433) 145,185 Wallis V, see Davies JS (131, Weinman E J, see Knight TF White A, see Zissblatt M 132) 119,173 90,108 (558) 137,190 Walsh JB, see Thong YA (498) Weismann G, Thomas L (534, White RG, see Askonas BA 157, 187 535) 157,189 (19) 117,169 Waltman SR, Brude RM, Weismann JL (533) 125,126, Whitehead RH, see Fox CA Benios J (583) 152, 191 189 (195) 146,175 Walton M, Fozzard HA 15, 74 Weit~ C, see Adam KR 10, 64 Whitfield JF, McManus JP, Wang CM, see Narahashi T Weiss Y, see Papanicolaou N Rixon RH (539) 150, 157, 11, 70 97, 110 189 218 AuthorIndex

Whitfield JF, see McManus Wioland M, Sabolovic D, Burg Yanai M, see Kakisawa H JP (333,334) 144, 158, C (545) 125,189 6, 68 162, 181 Wira C, Munck A (546) Yarger WE, see Harris RH Whitfield JF, see Rixon RH 156,189 92, 107 (431) 185 Wistar R, Hildeman WH (547) Yaron T, see Schlesinger M Wiederholt M, see Khuri RN 148,189 (459) 186 91,108 Witkop B, see Daly J 37, 66 Yasunobu KT, see Tanaka M Wiesner K, Kao W, Jay EWK Witkop B, see Tokuyama T 6,73 6,74 5,73 Yates FE, Urquhard J, Herbst Wigzell H, see Goldstein P Wittenberger C, see Pora E AL (550) 160,189 (233) 177 (420) 141,184 Yates FE, see Barger AC Wijemans P, see Astaldi A Wolf H, see Kaufmann R 76,103 (20) 159, 169 18, 68 Yazikof E de, see Scholz H Wijemans PS, see Astadi G Wolgast M, see Kallskog O 61,72 (21) 143,169 87,108 Young DA (551) 157,189 Wilkes B, see Aisenberg AC Wong FM, Taub RH, Sherman Young DA, see Mosher KM (7) 133, 134, 168 JD, Dameshek W (548) 189 (376,377) 157,182, 183 Williams AF, see Hunt SW Wood EH, HHeppner RL, Weld- Young DA, see Norden SK (262) 126,178 mann S 34, 74 (391) 157,183 Williams H, see Munro AJ Wooten GF, see Thoa NB Young RH, Hopkins P, Esparza (384) 124, 183 26, 73 A (552) 120, 189 Williams K, see Azar NA (24) Wortis HH, Nielsen S, Owen Yunis EJ, see Stutman O 117, 169 JJ (549) 117,133,189 (485-487) 133,134, 140, Williams RD, see Allen HL (9) Wortis HH, see Raft MC (426) 187 155,168 125,184 Yunis L, Hillgard HR, Marti- Williams RD, see Ellison EH Wright FS, see Davis BB nez C, Good RA (553) 134, (170) 155,174 89, 105 189 Williamson A, see Kreth HW Wright FS, see Johnston CI (303) 180 96,107 Zaalberg OBVD, Maul VA, Williamson HE, see Strandhoy Wright FS, see Knox FG Twisk MJ (554) 189 JW 101,112 84, 96, 108 Zahnd GR, Nadeau A, Mumlu- Willis LR, see Knox FG Wright FS, see Schneider EG dahl KE (555) 164, 190 83, 90, 108 101,111 Zatz MM, see Goldstein AL Witlis LR, see Schneider EG Wright FS, see Schnermann J (222) 136,177 80, 111 94,111 Zetterstrom R, see Aperia A Willis-Cart J J, St Pierre RL Wu CH, see Low PA 12, 69 99, 103 (540) 165,167,189 Wunderer G, Eulitz M 6, 74 Zijlstra J J, see Krisbeek AM Wilson DB (541) 120,189 Wunderer G, Fritz H, Wachter (307) 134, 180 Wilson DB, Billingham RE E, Machleidt W 6, 74 Ziller K (556) 1.25,190 (542) 120, 189 Wunderer G, see B6ress L Zimmerman JH, see Kupchan Wilson DR, Sonnenberg H 6, 65 SM 4, 69 92, 113 Wunderer G, see Romey G Zinkernagl RM (587) 131, Wilson R, Sj6din K, Bealmar 11,72 191 M (543) 117,189 Wunderlich P, see Kuschinsky Zinkernagl RM, Althage A Winchurch R, Ischizuka M, W 83,108 (588) 131,132, 191 Webb D, Braun W (544) Wunderlich P, see Schnermann Zinzar SM, Svetmoldavski GY 189 J 94, 111 (557) 117,190 Winchurch R, see Braun W Zipes, Mendez 21 (75) 171 Yakir Y, see Kook AI (301) Zissblatt M, Goldstein AL, Windhager EE, Lewy JE, 138,180 Lily F, White A (558) 137, Spitzer A 82,113 Yamabe H, see Alexander RW 190 Windhager EE, see Giebisch G 99, 103 Zlotkin E, Miranda F, Rochat 91, 106 Yamada K, see Shimizu T H 44, 74 Windhager EE, see Lewy JE 17, 48, 49, 73 Zollinger RM, see Ellison EH 82, 109 Yamagishi S, Sano T 16, 74 (170) 155,174 Windhager EE, see Spitzer A Yamamoto I, see Plescia O Zucker A, see Obika LFO 82, 112 (418) 158,184 101,110 Yanagihara K, see Noma A 16, 42, 71 Subject Index

aconine 6 arrhythmogenic effect of batrachotoxin 37 aconitine, action on Na channels 10 - - coral toxin 50 -, cardiac actions of 3, 39-42 arteriovenous fistula 93 -, chemistry of 5 aspirin and natriuresis 97 activation of Na channels 7, 8, 11, 16 ATP 52

- variables 8 atrium, veratrine actions 19, 20 active state of contractile elements 57 ATXII, see sea anemone toxin action potential, prolongation of 38, 39, 41, azathioprine test 142 47, 48, 49, 50 adaptation syndrome 148 batrachotoxin 5, 59 adenohypophyseal deficiency 163 -, action on Na channel 9 adenohypophysis and immune response 153, , on node of Ranvier 9, 10 154, 160 , arrhythmogenic action 37 adrenal cortex and immune response -, binding of 39 145-149 -, cardiac actions 37-39 and thymectomy 159 -, lethal dose 37 - deficiency 149 B cell precursors 140 adrenalectomy 146 - cells and thymosin 137 adrenaline 60, 61 - lymphocytes 126,129 adrenergic nerve fibres, intracardiac 31 benzocaine 22 - pathways 98 benzopyrene tumor 133 #-adrenoceptor agonists 58 Bezold-Jarisch effect 3 after-depolarization 8, 17, 18, 28, 29, 40 blood flow, intrarenal distribution of 77-79 age-related natriuretic response 100 , medullary 77 aldosterone and immunity 147 , renal, and vasodilators 76

- secretion and natriuresis 95 bradycardia 44 aldosteronism, primary 95 alimentary factors and immune functions Ca ++ channels 12

165 - concentration, intracellular 35 alloantigenic markers 130 - influx 46 allograft rejection 117,120,123,124,133, - and Na channels 14 135,137,139,140,141,146,147,150, -, oscillatory release 18

151,155 - uptake by sarcoplasmicreticulum 23, 24 p-aminohippurate, extraction ratio of 77 cAMP, see cyclic AMP anthoplettrin A 4, 6 cancer development 144, 145 -, cardiac actions of 48, 49 carbachol on sinoatrial node 16 -, kinetics of Na channels 12 cardiac action potentials 15-18 antibody production 117, 118, 135,137, - cells in culture and ATXII 47

145,146,150,155 - - -, ceveratrum actions 36, 37 antidiuretic hormone and natriuresis 97 - - -, scorpion toxin actions 45,46 antigen binding regions 131 - preparations, Nai-Ca o exchange 53-56

- receptor 132 - reflexes 3,4

- recognition 131,132 cardioactive steroids, Na pump inhibition by -, Thy-1 129 55, 56 antigenic markers 126,129,138,141,143 castration 151 anti-growth hormone antiserum 162, 163 catecholamines and immune response 158 anti-lymphocyte serum 139 - and sodium excretion 99 antithymosin antiserum 137 - andTTX 32, 33 anti-Toxoplasma factor 123 cell interaction, immunological 124 arrhythmogenesis 37, 41 cevacine 9 220 Subject Index cevadine 5, 9, 28 fermcyanide technique 80 -, chronotropic effect 19 fetus, immunostimulant influence 153 ceveratrum alkaloids, action on Na channel 7 filtration fraction 78, 79 , cardiac actions of 19 ft. 9 a-fluorohydrocortisone 94, 95 , chemistry of 4 folio acid deficiency 165 , circulatory effects 4 fractionalreabsorption 83, 84, 85, 91 , pharmacology of 3, 4 , positive inotropic effect 24, 25, 29 ft., gastrointestinal Na receptor 101 60 gating mechanism 7 choline 8 germine 5, 8 clonal selection theories 130 germine-3-acetate 9 cobalt 54 -, chronotropic effect 19 collecting duct, dilution in 91 germitetrine 25 concanavalin A 121,125,143 germitrine 5, 58 coral toxin 6, 12, 50 - on action potential 28, 29 cortical nephrons, sodium delivery from 92 -, positive inotropic effect 24, 29 ff., 60 corticosteroids and immune response 145 ff., - andTTX 32, 33 156,157 germ-line hypothesis 132 -, nuclear receptor for 156 GFR and fluid expansion 79, 80, 81

- and nucleic acid synthesis 157 -, intrarenal distribution of 80

- andprotein synthesis 157 glomerular filtration rate 94

- and thymus 160 and sodium excretion 79 corticotropin 140 - -tubular balance 90 - and immune response 155,156,157 gonads and immune response 151-153 cortisol 146, 148 goniopora toxin 6, 12, 50, 61 cross-circulation experiments and sodium graft immune reactions 120,121,123 excretion 96 graft-vs-host reaction 117 cyclicAMP 58,124,141,157,158 - - test 139,140,143 increasing agents 60, 61 growth hormone and immune response 155, - nucleotides in myocardial cells 36 156,162 eyclophosphamide 122 - - receptors 162 cytolytic activity 120 gryanotoxins 6 cytostatic activity 120 -, action on Na channel 10 cytotoxic activity 121 -, cardiac actions 42, 43 - cells 127 guanethidine 99

D 600 54 Hanssen'stechnique 79, 81 deoxycorticosterone 89,146,147 heart failure and Na excretion 93 deoxynucleotidyl-transferase 137,141 -, membrane currentsin 15-18 dihydro-ouabain 58 helper cell differentiation 124 -, inhibition of the Na pump by 61 - cells 122, 127,129,131 -, positive inotropic effect 60 - factors 131 -, potentiation of veratridine 31 hemagglutinin 151

- and TTX 32 - production 154 differentiation markers 129 hematocrit and sodium excretion 84 diterpenoids 6 hemodflution 78 DOCA escape 93 hemolysin production 118,146 - -saline treated rats 94 Henle loop and natriuresis 90, 91 dopamine &hydroxylase 99 hepatorenal syndrome 101,102 dye-dilution technique 77 h gate 7, 11 dwarf mouse 118,163 histamine 58 hormonal action in immune response 156 ff. estradiol 151, 152 - factors, influence on sodium absorption ethacrynic acid 89 94-99 extracellular fluid, volume expansion, chronic - interactions 159 93 hydrostatic pressure, capillary 84, 85 , , and natriuresis 87, 89 , peritubular and Na absorption 85, 86 , , and phosphaturia 98 , serosal 85 , , response of perinatal kidney to hypernatremia 89 99, 100 hypersensitivity, delayed-type 129,130 , , and sodium xeabsorption 90, 91, hypophyseal hormones and immune response 94, 95 154-156 Subject Index 221 hypophysectomy andthymus 162 major histocompatibility complex 129 antigens 131,132 IgG antibodies 146 manganese 54 IgM antibodies 123 mediator substance for immune reaction immune response and adrenal cortex 145 fL, 124,130 156,157 mesaconitine 40 - -, alimentary influences 165 metopyrone 148 - -, cell-to-cell interaction 118,119,123, m-gate 7, 9, 20 124 microcatheterization studies 92 and gonads 151 153 microsphere techniques 78 - -, hormonal coordination in 166,167 mineralcorticoids 93 - -, hormone mechanisms in 156 ff. - and sodium excretion 95 and hypophysis 153-156 myoblasts 45, 47

- -, organization and generation of 127 ft. andthymus 117 ft. Na ÷ see also sodium and thyroid 150,151 Na-Ca exchange 18, 35, 51 ff.

- system, cellular communications 130 - -, sarcolemmal 57 immunodeficiency 117 Na-K-ATPase and ceveratrum alkaloids 36 immunostimuiant effect 145 natriuresis 80, 82, 83,101 immunotolerance 119 - of hypertensive patients 85 inactivation gate 40 -, see also sodium excretion

- of Nachannel 7, 8, 11, 16 natriuretic hormone 95, 96

- time constant 20 neuroblastoma cells 13, 14 indomethacin and kidney 97 neuromuscular block 141 inert-gas washout 77 nucleic acids and cortisol 157 inotropic effect, positive 24, 25, 29 ff., 39, 42, 43, 44, 46, 48, 49, 56, 60 oncotic pressure and sodium absorption , , chain of events 63 82, 83, 84, 85 - -, -, presynaptic 31 oscillation of membrane potential 28 interstitial pressure and Na absorption 86 oscillatory after-potentials 18, 41 ionic selectivity, Na channels 9, 10 ouabain 53, 54 isometric contraction 57, 58 ovalb umin 155 jerveratrum alkaloids and alkamines 4 pacemaker cells, hyperpolarization of 16 juxtameduUary nephron function 92 palytoxin 59 parathyroid hormone and natriuresis 97, 98 K ÷ accumulation 28 perinatal kidney, responses of 99 -, extracellular and inotropy 61 peritubular capillary, permeability surface killer ceils 129 area 87 killing assisting factor 124 phosphatase, alkaline 125 klinotropic effect, positive 23, 39, 42, 43, phosphaturia 98 46, 49 phosphodiesterase inhibitors 58 phytohemagglutinin 125, 143 lldocaine 14 plaque-forming cells 121,123, 126,133, llpopolysaccharides 124 137,149,150 lithium ions 8, 20, 54 - cellassay 117,118 liver and sodium reabsorption 101 plasma cells 130 lymph nodes, thymus-dependent zones 125 - protein and natriuresis 82, 83, 84 lymphocyte differentiation markers 129, 130 polyomavirus 117,134 - stimulating hormone 143,144 polypeptide toxins 6 lymphocytes, see also B and T cells polyribonucleotides 158

- in bone marrow, see also B cells 126 pregnancy and immune response 153 -, corticoidreceptors 157 pressure, oncotic, peritubular 78 - and cyclic AMP 158 procaine 22 - and growth hormone 162 progesterone implants 152 -, immunological functions of 128, 129 propranolol 26 -, influence of adrenocortical hormones on -, effect on Na channels 43, 62 146 ff. prostaglandin synthesis inhibitors 158 -, intracellular enzymes 125 prostaglandins and sodium excretion 97 lympholine synthesis 157 prothymocytes 141 lymphotoxines 124 protoveratrine 25, 28, 30, 37 lysosomal enzymes 157 proximal tubule, altered permeability of 84 222 Subject Index

proximal tubule, sodium reabsorption in 83 - - and BTX 38

Purkinje fibres, aconitine actions 40 - - in cardiac action potential 15-18

- -, batrachotoxin action 37, 38 - -, effects of toxins on 3

, veratrine actions 21, 22 , excitation-concentration coupling, role puromycin 157 in 50, 51

pyridoxal deficiency 165 - - and grayanotoxin 43 , inactivation of 49

Ranvier node 7, 10, 11, 20 - - and scorpion toxin 45 reabsorption coefficient 87 , silent 12, 13,48 refractory period 48, 49 , TTX-insensitive 13 relaxation time 23, 57, 58 -concentration, intracellular 31, 34, 35 renal sodium absorption, see under sodium - excretion, age-related 100

76 - - and anesthesia 84 renin-angiotensin system 93 , gastrointestinalcontrol of 101,102 repolarization, delayed 17 - - and renal blood flow 76, 77 -, early 17 -, extracellular and inotropy 60 reserpine 44, 47 -, fractionalreabsorption 83, 84, 85, 93 RNA 124 - inactivation 44,47

- polymerase activity 157 - ion receptor 101 rosette-forming ceil test 117 - influx and positive inotropic effect 63, 64

- cells 137, 138, 142 - ionsand Ca 2+release 51 - permeability changes 7, 8 saline infusion 76, 77 - in plasma and natriuresis 88, 89 sarcoma graft rejection 145 - pump 36, 52 - rejection 137 inhibition 56, 61 saxitoxin 2, 13,58 , stimulation of 43 scorpion toxins 6, 54 - reabsorption, micropuncture studies - -, action on Na channel 10, 11 91, 92 - -, action on nerve fibres 10 - -, proximal 78 - -, cardiac actions of 44-46 - -, segmentaltubular 90-93

- -, presynapticaction of 59 - retention, chronic model 93 sea anemone toxins 6, 46, 61 spinal cord section and natriuresis 98

- - -, action on Nachannel 11, 12 spironolactone 147

- - -, cardiac actions 46-49 spleen cells, killer activity 146 secretin 77 - -, transfer of 134 single nephron glomerular filtration rate - extracts 137 80, 81, 94 Starling forces 86 sinoatrial node, effects of ceveratrum 19 stress and immune response 148, 149 sinus tachycardia 19 superficial nephron, filtration fraction 79 slow inward current 15, 61 suppressor T cells 121,127,129,130 sodium absorption, dependency on plasma Na 88 -90 T cell replacing factor 131 and glomerular filtration rate 79-81 - cell-B-cell interaction, mechanism of , hemodynamics influencing 76-79 122-124, 130, 131

- -, intestinal 84, 85 - cells, antigen recognition 131 - -, neurohumoral effects upon 98, 99 and B cells, distinction of 125,126 - -, physical factors influencing 82-90 cortisol-resistant 126 - accumulation, intracellular 54, 56 immunocompetent 127

- channel-gate toxins, see also individual immunological functions 128, 129 toxins invasion of 133 antagonists to 13, 14 killer 129 arrhythmias produced by 18 maturation of 126,127, 143 cardiac action of 15 ft. secretion of mediator substance 123 and cardiac reflexes 3, 4 subpopulations in 125, 126, 127 chemistry of 4-6 testosterone 151,152 competitive interactions 14 tetracaine 14 definition of 3 tetrodotoxin 2, 8, 12, 13, 15, 16, 19, 20, 28, and isometric contraction 57 47, 48, 49, 50

kinetics of Na channels 13 - and batrachotoxin action 38 mode of action of 7 ff. , blockade of sarcolemmal Na channels by receptors for 13 58, 59

- channels aconitineaction 41 - and development 36 Subject Index 223

- andgrayanotoxin 42,43 tubular surface area 90

- insensitive Na channel 36 tubule, distal, role in natriuresis 90 ff.

-, mode of action 31 - geometry and sodium reabsorption 90 -, negative inotropic action 18 tubuloglomerular feedback 94 and Purkinje fibres 22 tumor development 144, 145 -, veratridine antagonism 32, 33, 34 tumors, experimental 117 theophyUine 58,60,61 thymectomy 147 urine reinfusion 92 -, effects on lymphocytes 125

- and hormones 159 vasopressin 89 -, immunological consequences of 117 vena cava obstruction 93 -, neuromuscular transmission in 141 ventricular muscle, aconitine actions 40, 41 -, substitutive therapy for 133 ff. and batrachotoxin 38, 39 thymic culture supernatant, injection of 134 -, veratridine actions 23 ff.

- extracts 135 ff. - myocardium, action potential 15

- factor in serum 142 veracevine 5, 8, 24

- grafts 133,134 veratridine 5, 11, 58

- hormone 155 , action on Na channel 7 ff

, homeostatic 140,141 - and Ca ++ 14

- humoralfactor 138,139,140 - and Cainflux 53, 54 - hypertrophy 149 -, cardiac actions 19ff. - nurse cells 133 - in cultured cardiac cells 36 thymin 141 -, mechanism of inotropic effect 29 ft. thymocytes 127 - and noradrenaline release 26

-, transfusion of 134 - in pancreas 12 thymopoietin 141,159 - on Ranvier node 7 thymosin, actions of injected 136 ft. -, reserpine-like effect 26

-, activity tested in vitro 138 - and silent Na channels 12 -, amino acid sequence of 136 - andTTX 32, 33, 34 thymosterines 145 - and ventricular action potential 26, 27 thymus-adenohypophysis interaction 162 ff. veratrine 4

- -adrenalinteractions 161,162 -, action on action potential 19 -, generation of antigen diversity 132 -, actions onatrium 19-21 -, lipidic extracts from 144, 145 -, positive inotropic effect 20, 22, 23 ff., 60

-, morphology of 128 - relaxation time 57 -, peptitic extracts from 136-144 -, ventricular actions 23 ff. -, polypeptide fraction 144 veratrum alkaloids 4 -, role of, in immunity 116 ff., 149,154 volume expansion, extracellular 76, 77, 84, , , inimmunotolerance 119,120 85, 86 - -thyroid interactions 161 see also under extracellular thyreotropin 156,163 thyroid hormone and sodium transport 98 Walker sarcoma grafts 145

- and immune response 150, 151 washout hypothesis, medullary 78 thyroxine 163,164 wasting 133,139,140 injection 150,151,154 transmitter release 11, 26 zygadenine 24 tuberculin 150