ABSTRACT
The Formation and Release of Surplus Acetylcholine by a Sympa the tic Ganglion
Howard S. Katz
Dept. of Physiology
McGill University
The formation and release of surplus acetylcholine was studied in perfused sympathetic ganglia of cats. Surplus acetylcholine is the extra acetylcholine
that accumulates in the presence of choline and an anticholinesterase. Surplus
acetylcholine formed rapidly when eserine was the anticholinesterase, but slowly when neostigmine or ambenonium was used. Chronically decentralized ganglia
synthesized no surplus acetylcholine. These experiments suggested that surplus
acetylcholine was stored presynaptically and the results were discussed in
relation to the location of acetylcholinesterase. Surplus acetylcholine was 3 labelled by perfusing resting ganglia with es erine- H-choline-Locke. Subsequent
stimulation of the preganglionic nerve failed to release radioactivity although \ unlabelled acetylcholine was released. These experiments~ suggested that surplus
acetylcholine did not mix with releasable transmitter. Perfusion with high-K+
or acetylcholine or carbachol injections effectively released surplus acetyl-
choline, but the released acetylcholine did not augment the ganglion-stimulant
effect of carbachol. These results were discussed in relation to the synthesis
and storage of acetylcholine. --; ..'
Short Title
FORMATION AND RELEASE OF SURPLUS ACETYLCHOLINE THE FORMATION AND RELEASE OF SURPLUS ACETYLCHOLINE BY ASYMPATHETIC GANGLION
by
Howard S. Katz
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science.
Department of Physiology, McGill University, Montreal. April, 1970.
~,:: t. 1'::\ ~ ~ Howard S. Katz ir,' 1970 Dedicated te Dara-Carelyn. ACKNOWLEDGMENTS The problem discussed in this thesis was suggested by my research director, Dr. Brian Collier, Assistant Professor in the Department of
Pharmacology and Therapeutics, McGi~l University. It is a pleasure for me to record my appreciation for the assistance and guidance afforded me by
Dr. Collier, both during the course of the research, ~ld during the preparation of this thesis. His des ire to transform students into thinking scientists is acknowledged by those of us who have had the opportunity of working with him.
l would also wish to thank Dr. David Bates, Chairman of the
Department of Physiology, for the opportunity of pursuing my research as a registered student in his department. In this respect l am thankful to
Dr. Mark Nickerson, Chairman of the Department of Pharmacology and Therapeutics, and the rest of the members of his staff, particularly Mrs. Mary Robertson, for the opportunity of completing my research in Physiology by working in their department. The members of the Departmeüt of Pharmacology and Therapeutics have been extremely generous and helpful, for which l am sincerely appreciative.
l would also wish to thank Miss Celia Lang for her assistance in the
course of these expetiments, and for teaching me some of the procedures used;
Mr. Peter Poon and Mr. Bruce Harfield were of assistance during the latter
part of the research.
My special thanks are due to my mother for diligently typing the rough
version of this manuscript and to both my parents for their consistent
encouragement thro~ghout my studies. l have also received moral encourage
ment from afar from L.S.; l am particularly thankful to her for aiding me in
overcoming certain obstacles during the course of the research. l am very grateful to Dr. Daya Varma, Associate Professor in the
Department of Pharmacology and Therapeutics, and Mr. Saleh Saleh, fellow graduate student, for constructively criticizing the rough manuscript.
The care and skill of Mrs. Tyleen Katz in typing the final manuscript, while burdened with normal departmental work, is gratefully acknowledged.
Her warm understanding has strengthened my conviction that British girls are super.
The figures presented in this thesis vere drawn carefully by
Mr. Gordon Boyd, and photographed with precision by Mr. Royal Raymond.
This work was supported by a grant from the Medical Research Council of Canada. TABLE OF CONTENTS
PREFACE • • • • • 1-3
I. INTRODUCTION • 4-25
A. Acetylcholine as a Neurotransmitter in the Peripheral Nervous System • • • • • • • • 5-7
B. Storage of Acetylcholine 8-12
C. Pools of Acetylcholine in Nervous Tissue 12-14 a) Superior cervical ganglion of the cat 12-13 b) Mammalian brain • • • • 14
D. Synthesis of Acetylcholine • • • • • 14-20 a) Coenzyme A and active acetate • • • • 14-15 b) Choline • • • • • • • • • • • •• • • • • • . 15-18 c) Choline acetyltransferase and the subcellular location of acetylcholine synthesis • • • • • • • • • • • • 18-20
E. Acetylcholinesterase and the Fate of Released Acetylcholine 20-25 a) Neuronal barriers to cholinesterase inhibitors • • • • • 21-22 b) Functional and residual AChE • • • • • • • • • • • • • • 22-24 c) The physiological role of AChE in the superior cervical ganglion • • • • • • • • • • 24-25
II. METRODS AND MATERIALS 26-39 A. Ganglion Perfusion . . . 27-29 B. Experimental Procedure • • • • • • 29-30 a) Formation of surplus acetylcholine • • • • • 29 b) Labelling of surplus acetylcholine • 29-30 c) Release of acetylcholine into different anticholinesterase containing Locke solution • • • • • • • • • • • • • • 30 C. Chronic Decentralization of Superior Cervical Ganglia • 30-31
D. Stimulation of the Preganglionic Sympathetic Nerve 31 E. Ganglion Extracts • 31-32
F. Bio-assay 32-35
G. Separati~n of Labelled Material • • 35-37 a) Separation by gold chloride 35 b) Separation by reineckate • • • • 35-37 H. Acety1ation of 3 H-Cho1ine 37
I. Measurements of Radioactivity 37-38
J. Materia1s 38-39
K. Statistica1 Ana1ysis 39
III. RESULTS 40-76
A. Formation of Surplus Acetylcholine in the Presence of Various Anticho1inesterase Agents . • • • • • • • • 41-45
B. Re1ease of Acetylcholine by Nerve Stimulation into Different Anticho1inesterase Agents • •••••• • • . • • • 46
C. Surplus Acetylcholine Formation in the Chronica11y Decentra1ized Ganglion • • • • 46-48
D. Labe11ing of Surplus Acetylcholine 48-52
E. Fai1ure of Nerve Stimulation to Re1ease Surplus Acetylcholine: Perfusion with Eserine-Choline-Locke • • • • • • • . . . • • • • 52-58
F. Fai1ure of Nerve Stimulation to Release Surplus Acetylcholine: Perfusion with Eserine-Hemicho1inium-3-Locke 58-63
G. Release of Surplus Acetylcholine by High K+-Locke • 64-67
H. Re1ease of Surplus Acetylcholine by Acetylcholine or by Carbacho1 67-73 a) Re1ease of surplus acety1choline by acetylcho1ine 67-70 b) Re1ease of surplus acetylcholine by carbachol 70-73
1. Ganglion Stimulation by Carbachol in the Presence or Absence of Surplus Acetylcholine . . . . 73-76 IV. DISCUSSION ...... · · · · · 77-89 A. Formation of Surplus Acetylcholine · · · · · · · · · 78-79 B. Turnover of Surplus Acetylcholine . . · · · · · · · · · 80-81 C. Release of Acetylcholine by Nerve Stimulation 82-85 D. Release of Surplus Acetylcholine by K+ . . . . · · · · · · · · · 85··86 E. Release of Surplus Acetylcholine by Acetylcholine or by Carbacho1 86-89 V. SUMMARY · · · · · 90-92 VI. BIBLIOGRAPHY · · · · · 93-106 LIST OF TABLES
Table
l Formation of surplus ACh by ganglia perfused with 44 neostigmine-Ch-Locke
II Formation of surplus ACh by ganglia perfused for 90 min 45 with various anti-ChE agents
III Accumulation of surplus ACh by normal and chronically 49 (1 week) decentralized ganglia during perfusion with eserine-Ch-Locke for 60 min
IV The labelling of surplus ACh in superior cervical ganglia; 51 ganglia were perfused at rest for 60 min with eserine- 3H-Ch-Locke
V The effect of nerve stimulation upon the efflux of radio- 55 activity and upon the release of ACh from superior cervical ganglia perfused with eserine-Ch-Locke; the ganglia's surplus ACh pool had been labelled
VI Labelled and unlabelled ACh in superior cervical ganglia 56 that had been perfused for 60 min with eserine-3H-Ch- Locke and then for 60 min with eserine-unlabelled Ch- Locke
VII The effect of nerve stimulation upon the efflux of radio- 61 activity and upon the release of ACh from superior cervical ganglia perfused with eserine-HC-3-Locke; the ganglia's surplus ACh pool had been labelled
VIn Labelled and unlabelled ACh in superior cervical ganglia 62 that had been perfused for 60 min with eserine-3H-Ch Locke and then for 60 min with eserine-HC-3-Locke
IX The effect of high K+ perfusion (6 min) upon the efflux 66 of radioactivity from superior cervical ganglia whose surplus ACh pool had been labelled
X The effect of ACh perfusion (6 nd.n) upon the efflux of 69 radioactivity from superior cervical ganglia whose surplus ACh pool had been labelled
XI The effect of injected or perfused (4 or 6 min) carbachol 74 upon the efflux of radioactivity from superior cervical ganglia whose surplus ACh pool had been labelled LIST OF FIGURES
Figure
1 A typica1 bio-assay for the estimation of ACh extracted from a cat's superior cervical ganglion 34
2 Rate of increase of ganglion ACh content in gang1ia with different anti-ChE agents 42
3 The re1ease of ACh from perfused superior cervical gang1ia during pregang1ionic nerve stimulation in the presence of different anti-ChE agents 47
4 Fai1ure of nerve stimulation to re1ease surplus ACh during perfusion with eserine-Ch-Locke; the gang1ion's surplus pool had been 1abe11ed 53
5 Turnover of surplus ACh 57
6 Fai1ure of nerve stimulation to re1ease surplus ACh during perfusion with eserine-HC-3-Locke; the gang1ion's surplus pool had been 1abe11ed 60
7 The re1ease of surplus ACh by high K+ -Locke 65
8 The re1ease of surplus ACh by ACh 68
9 The re1ease of surplus ACh by ACh in the presence of different anti-ChE agents 71
10 The re1ease of surplus ACh by carbacho1 72
11 Contractions of the cat's nictitating membrane induced by carbacho1 injections in the absence and presence of surplus ACh 76 1 e·
PREFACE 2
This thesis is a report of work which studied the formation and re1ease of "surplus" acetylcho1ine (ACh) in the catIs superior cervical ganglion. Surplus ACh is the extra ACh that is synthesized and stored by a ganglion when it is exposed to an anticholinesterase (anti-ChE) agent and is provided with a supp1y of choline (Ch) (Birks and MacIntosh,
1961), Little is known about this surplus ACh; previous work had shown on1y that it can exist, and that its accumulation does not immediately increase the amount of ACh re1eased by nerve impulses. The present study examined the formation of surplus ACh in decentra1ized ganglia, and attempted to corre1ate the rate of formation of surplus ACh in normal gang1ia with the physica1 properties of the anti-ChE agent used; these resu1ts contribute to knowledge about the site of storage of surplus ACh.
In addition, the rate of exchange (or mixing) of surplus ACh with the normal transmit ter store was measured; these resu1ts provide information about the physio1ogica1 significance of surplus ACh formation and about
the process of synthesis and storage of transmitter ACh. Fina11y, various
procedures were tested for their abi1ity to re1ease surplus ACh and the
experiments showed that the re1ease of surplus ACh and the re1ease of
transmitter (depot) ACh can be quite distinct; these resu1ts add to our
know1edge about the storage and re1ease of ACh at cholinergie synapses.
This thesis begins with a review of the 1iterature pertinent to its
subject; this is not a comprehensive review of ACh as a synaptic trans
mitter because such 1iterature is extensive and has been reviewed on
numerous occasions (see e.g. Hebb .and Krnjevié, 1962; MacIntosh, 1963;
VoIle, 1966), The methods used in this investigation are then described;
the results are presented and discussed in relation to relevant work by 3
others. The experiments, except where 1t 1s stated expl1citly, are original as far as the author is aware. 4
I. INTRODUCTION 5
A. Acetylcholine as a Neurotransmitter in the Peripheral
Nervous System
Claude Bernard's early experiments (1856) on the effect of curare on impulse transmission from nerve to skeletal muscle in the frog clearly demonstrated that a drug can Interfere with the process of neuromuscular transmission without having a direct effect on the nerve or on the muscle.
These experiments provided the first clear evidence that there might be a specialized neuro-secretory process,although Du Bois-Reymond (1875) had previously suggested the possibility of chemical transmission. The demonstrations of Langley (1901) and Elliott (1904, 1905) that the effects of injected adrenaline paralleled those of sympathetic nerve stimulation, led Elliott (1904) to suggest that adrenaline itself is the substance released by sympathetic nerve stimulation. An analogous suggestion by
Dixon (1906, 1907) equated the action of "inhibitin", a muscarine-like substance, to stimulation of the parasympathetic nervous system. Eight years later, Dale (1914) made a detailed study of the pharmacological actions of acetylcholine (ACh) and showed that the effects of injected ACh
(in small amounts) were very similar to the effects of stimulating para- sympathetic nerves. Dale also observed that when this parasympathomimetic activity (or "muscarinic" action of ACh, as he called it) was abolished by atropine, another action of ACh was revealed which closely resembled
that of nicotine as a stimulant of autonomie ganglia and skeletal muscle.
It was, however, the ele~ant experiments by Loewi (1921) on the frog's
heart which first provided unequivocal evidence for neurohumoral trans
mission. He showed that stimulating parasymapthetic cardiac nerves
released an ACh like substance which he termed "Vagusstoff". This 6
materia1 affected a second denervated heart as if its own parasympathetic nerve had been stimu1ated, and it was shown 1ater that Vagusstoff had
ACh-1ike effects on other tissues, such as the frog's stomach (Brinkman and van Dam, 1922). Loewi and Navrati1 (1926~, 1926b) demonstrated the instabi1ity of Vagusstoff in a1ka1i, and the reappearance of activity on subsequent acety1ationof the a1ka1i-destroyed materia1; atropine cou1d a1so prevent the action of Vagusstoff on test preparations.
The demonstration that horse spleen con tains large amounts of ACh
(Dale and Dudley, 1929) 1ed others to study the distribution of choline esters in the mamma1ian nervous system. Chang and Gaddum (1933) iso1ated
ACh from horse sympathetic gang1ia, and Feldberg and Gaddum (1934) showed the re1ease of ACh from the catis superior cervical gan~lion when its pregang1ionic nerve was stimu1ated; Feldberg and Gaddum used the technique of ganglion perfusion deve10ped by Kibjakow (1933). The substance re-
1eased from the active ganglion was identified as ACh by a series of para11e1 bio10gica1 assays, by its ease of destruction by a1ka1i, by the abi1ity of atropine or curare to b10ck its effects on appropriate test organs, and by the fact that the presence of eserine in the perfusion f1uid was essentia1 to preserve the re1eased ACh. Antidromic activation of the ganglion was ineffective in releasing ACh (Feldberg and Vartiainen,
1934), and injection of exogenous ACh to the superior cervical ganglion was shown to stimu1ate the ganglion (Feldberg and Vartiainen, 1934;
Paton and Perry, 1953). Thus the evidence that ACh is a transmit ter
substance in sympathetic gang1ia is fair1y complete; the evidence that ACh
is a neurotransmitter in parasympathetic gang1ia is most1y indirect (Perry
and Ta1esnik, 1953). 7
The observation that ACh was a transmitter substance at post ganglionic parasympathetic nerve endings and at autonomic gang lia was extended to the neuromuscular junction when Dale, Feldberg and Vogt
(1936) released ACh from perfused skeletal muscle during motor nerve stimulation, and Brown, Dale and Feldberg (1936) showed that normal skeletal muscle contracted when small doses of ACh were injected close arterially.
The evidence that ACh is a neurotransmitter was reviewed by Feldberg
(1945) who emphasized the following criteria: (a) the presence of ACh in certain tissues together with the enzymes for its synthesis and destruction; (b) the faithful reproduction of physiological events by properly applied exogenous ACh; (c) the potentiation of the effects of applied and released ACh by an anti-ChE agent; (d) the block of trans mission and of the action of ACh by certain drugs at specific loci;
(e) the release of ACh during nerve stimulation. These criteria are the
basic pre-requisites of a neurotransmitter (see e.g. Feldberg, 1945;
McLennan, 1963) and for the superior cervical ganglion ACh satisfies aIl
of them except (c). The poor potentiation by eserine of ganglionic
synaptic potentials (Eccles, 1944) is most easily explained by supposing
that the transmit ter action is terminated by diffusion of ACh from the
receptor with subsequent hydrolysis, rather than by immediate destruction
of the mediator by AChE (Ogston, 1955; Emmelin and MacIntosh, 1956); AChE
is located presynaptically CKoelle and Koelle, 1959) and therefore is
poorly placed for the Immediate termination of transmit ter action. 8
B. Storage of Acetylcholine
One of the important requirements that is expe~ted of a possible neurotransmitter is that it should be present, and this implies that the transmitter must be stored ready for use. Electrophysiological studies of ACh release at the amphibian neuromuscular junction first suggested that this mediator is stored in pre-formed, multimolecular packages
(Fatt and Katz, 1952). In these experiments, miniature end plate potentials (m.e.p.p.s.) were observed during recording of the transmembrane potential of resting nerve-muscle preparations. The m.e.p.p.s. were small randomly-occurring depolarizations that had aIl the characteristics of very small end plate potentials (e.p.p.s.) and each m.e.p.p. was thought to be the result of a synchronous dis charge of ACh from nerve endings.
The amplitude of m.e.p.p.s. was reduced by tubocurarine, potentiated by an anti-ChE and under normal conditions was always about the sàme size;
ACh produced a graded response when it was applied directly to the end plate of the muscle and thus m.e.p.p.s. must have been the result of multi- molecular discharges. The e.p.p. that could be measured when the nerve was stimulated was shown to be the sum of many synchronous m.e.p.p. discharges (Del Castillo and Katz, 1954; Boyd and Martin, 1956); a m.e.p.p. response, due to the action of many ACh molecules, represents the smallest unit or "quantum" release (Del Castillo and Katz, 1954).
About the same time, electron microscopie examination of different
synapses revealed the presence of small spherical structures that were
clustered near the terminaIs of the presynaptic membrane. These "synaptic vesicles" (De Robertis and Bennett, 1954) have been found in most synaptic
regions, including the neuromuscular junction (Reger, 1958), the central 9
nervous system (Palay, 1956; Gray, 1959a, 1959b), frog sympathetic ganglion (Taxi, 1961; De Robertis and Bennett, 1954, 1955), ciliary ganglion of the chick (De Lorenzo, 1960), and the superior cervical ganglion of the cat (Elfvin, 1963). It was suggested that these synaptic vesicles are the subcellular structunsin which ACh is stored and from which
ACh is released, a single vesicle is supposed to contain one quantum and its release gives rise to one m.e.p.p. (De Robertis and Bennett, 1955;
Del Castillo and Katz, 1956, 1957; Katz, 1962).
Evidence for the vesicular hypothesis is still not overwhelming, but no feasible alternative has been suggested and there is little evidence against it. Attempts to change the appearance or numbers of vesicles in synaptic regions by processes that alter transmitter turnover have been largely unsuccessful (Birks, Huxley and Katz, 1960; Birks, 1966). However,
Hubbard and Kwanbunbumpen (1968) and Jones and Kwanbunbumpen (1968) have demonstrated recently that bathing the rat phrenic nerve-diaphragm preparation in 20 mM K+ , or nerve stimulation in the presence of hemi- cholinium #3 (HC-3), significantly reduced the number of vesicles that could be counted on electron micrographs.
Electrical events similar to those recorded from the neuromuscular junction have been demonstrated in autonomie ganglia. Spontaneous miniature synaptic potentials have been recorded from frog sympathe tic ganglia (Nishi and Koketsu, 1960; Blackman, Ginsborg and Ray,1963a; Hunt and Nelson, 1965), their amplitude was reduced by tubocurarine and their frequency was increased by raising external K+ ; Ca* was required for release, and the action of this ion was antagonized by an elevated Mg* conce~tration. The excitatory postsynaptic potential recorded during 10
nerve stimulation was shown to De compob~d of multiples of the miniature junction potentia1 (B1ackman, Ginsborg and Ray, 1963b) as has been demonstrated for avian (Martin and Pi1ar, 1964) and mamma1ian gang1ia
(B1ackman and Purves, 1969).
The suggestion that ACh is stored in vesic1es was supported by the demonstration that synaptic vesic1es iso1ated from disrupted synaptosomes contain ACh (Whittaker, 1959; Gray and Whittaker, 1962; De Robertis,
Rodriguez de Lores Arnaiz, Sa1ganicoff, Pe11egrino de Ira1di and Zieher,
1963; Whittaker, Michae1son and Kirk1and, 1964; Whittaker and Sheridan,
1965). This biochemica1 approach has as yet on1y succeeded when app1ied to brain tissue and its re1evance to the process of neurotransmission in the mamma1ian periphera1 nervous system remains to be evaluated.
"Synaptosomes" (Whittaker et a1., 1964) are pinched off nerve endings that are forme"d when brain is homogenized in isotonic sucrose. These structures have a diameter of about 0.5 ~ and they contain synaptic vesic1es and sma11 mitochondria within their cytop1asm; about 80% of brain
ACh is contained in synaptosomes, the other 20% is presumed to be outside nerve endings. The synaptosomes can be disrupted by hypo-osmotic shock, but many of the vesic1es that they contain survive and can then be
separated from other materia1 by differentia1 centrifugation. These
purified synaptic vesic1es contain about 50% of the nerve ending ACh; the
rest of the ACh in synaptosomes appears to be free in the cytop1asm
(Whittaker, 1959; Whittaker et al., 1964; Beani, Bianc4i, Megazzini,
Ba110tti and Bernardi, 1969).
In vivo experiments have shown that vesicu1ar ACh can be 1abe11ed
by injecting radioactive Ch intracerebra11y to guinea-pigs (Chakrin and Il
Whittaker, 1969), but the procedure a1so 1abe11ed aIl other pools of
ACh and Chakrin and Whittaker have not yet tested the re1ease of
1abe11ed ACh. There is still doubt whether or not vesic1es can in- corporate ACh when synaptosomes are incubated with 1abe11ed Ch or with
1abe11ed ACh. Potter (1968) incubated synaptosomes with 1abe11ed Ch and found much of the synthesized ACh in vesic1es, but this was not so in the . . 14 experiments of Marchbanks (1968b, 1969). Marchbanks found that C-Ch was converted to 14C_ACh by synaptosomes but that on1y cytop1asmic ACh 14 was 1abe11ed; the rate of incorporation of C-ACh into vesic1es was 1ess than 10% of its rate of formation in cytop1asm. Marchbanks (1968a) a1so showed that 14C-ACh was taken up by synaptosomes, but again on1y cytop1asmic
ACh was 1abe11ed, and in his experiments puritied vesic1es (incubated at
SoC or 26°C) did not take up ACh. Burton (1964), however, had demonstrated previous1y the passive uptake of 14C_ACh into synaptic vesic1es, and its subsequent re1ease by the addition of un1abe11ed ACh, whi1e Kuriyama,
Roberts and Vos (1968) found synaptic vesic1es were able to bind not on1y
ACh, but Ch and GABA as weIl.
However, both Burton (1964) and Kuriyama et al. (1968) iso1ated the vesic1es from disrupted synaptosomes by the method of De Robertis et al.
(1963), and it is possible that their "purified" vesicu1ar fraction was
contaminated with intact synaptosomes (Whittaker et al., 1964); Marchbanks
(1968a) therefore suggested that the uptake of ACh seen by Burton and
Kuriyama et al. was into synaptosomes and not into vesic1es. However,
Guth (1969) iso1ated synaptic vesic1es by the method of Whittaker et al.
(1964) as modified by Barker, Amaro and Guth (1967) and demonstrated that
vesic1es can accumu1ate ACh within the first min of incubation at 37°C; 12
this uptake was against a concentration gradient.
C. Pools of Acetylcholine in Nervous Tissue
a) Superior cervical ganglion of the cat. Prolonged stimulation of the superior cervical ganglion of the cat in the presence of hemicholinium
#3 (HC-3) released only about 85% of the total extractable ACh of the ganglion (Birks and MacIntosh, 1961). HC-3 had no effect upon ACh release, but prevented the synthesis of the transmit ter by inhibiting the eutry of Ch into nerves (MacIntosh, Birks and Sastry, 1956; Gardiner,
1957; MacIntosh, 1961). Thus a measure of depletion in HC-3's presence was a measure of the ganglion's releasable pool of ACh. This releasable
ACh, which amounted to about 220 ng in an average ganglion was called
"depot" ACh by Birks and MacIntosh and was further divided into two sub fractions: a smaller pool, which contained transmit ter that was more readily released by nerve impulses, and a larger less readily releasable pool. The relationship between these two sub-fractions was not clear, but Birks and MacIntosh suggested that they exist in "series" such that
ACh from the larger must first pass through the smaller before being released. The recent findings that there seems to be a preferential release of newly-synthesized transmit ter in the superior cervical ganglion of the cat (Collier and MacIntosh, 1969; Collier, 1969) suggests that
"naw" ACh enters and is released from the more readily available sub fractioll before it can mix with the bulk of the depot pool. Those vesicles that are seen in electron microscopie studies to be closely aligned to the presynaptic terminaIs (Elfvin, 1963) may correspond to the readily releasable sub-fraction. 13
It is c1ear that depot ACh must be located in nerve termina1s but the 15% of the transmitter substance that is not avai1ab1e for
re1ease by nerve impulses, ca11ed "stationary" ACh by Birks and MacIntosh, may be located in the intraganglionic axon.
Birks and MacIntosh (1961) showed that another pool of ACh appeared when a resting ganglion was perfused with a medium containing a source of
Ch and an anti-ChE agent such as eserine; under these conditions the
ganglion synthesized and stored extra ACh which was ca11ed "surplus" ACh.
The accumulation of surplus ACh in the presence of eserine shows
that ACh was continua11y being made by the resting ganglion; in the
absence of an anti-ChE, this ACh was quick1y destroyed. The formation
of surplus ACh was dependent upon the presence of both an anti-ChE agent
and a source of Ch and its accumulation doubled the ACh content of
gang1ia within 1 hr.
Birks and MacIntosh (1961) presumed that surplus ACh was synthesized
in the nerve ending region, and the present experiments that measured
the formation of surplus ACh in chronica11y decentra1ized gang lia supp1ied
some evidence to support their suggestion. Surplus ACh must exist intra
ce11u1ar1y since it did not 1eak into the perfusate, and Birks and
MacIntosh suggested that surplus ACh might exist free in the nerve-ending
cytop1asm.
The re1ease of ACh by nerve stimulation from a ganglion that had
formed a full complement of surplus ACh was about the same as the re1ease
from a fresh1y eserinized ganglion and this suggested that surplus ACh
was not immediate1y avai1ab1e for re1ease. The present work was the
first to study mixing between surplus ACh and the re1easab1e ACh. 14
b) Mammalian brain. The subcellular distribution of ACh in mammalian brain tissue has been studied by numerous workers (see e.g.
Whittaker, 1959; De Robertis et al., 1963; Whittaker et al., 1964;
Mar chbanks, 1968b; Chakrin and Whittaker, 1969; Takeno, Nishio and Yanagiya,
1969). When brain was homogenized in eserine-sucrose, about 20% of total brain ACh appeared free in a high speed supernatant: this was called "free"
ACh and represented ACh that was outside nerve endings. "Bound" ACh was present in synaptosomes and appeared in two forms when the synaptosomes were broken by hypo-osmotic shock: a "stable" form, which was associated with the synaptic vesicles, and a more "labile" form found in the cytoplasm of the synaptosome. Drastic procedures were required to release stable ACh from vesicles, suggesting that there might be a chemical bond between ACh and the vesicle matrix; the ACh in vesicles was protected from AChE. Labile
ACh that appeared to be free in the cytoplasm was only recovered in the presence of an anti-ChE. This ACh in the nerve ending cytoplasm might be ana.logous to surplus ACh in the superior cervical ganglion of the cat, .and the present wotk allowed certain comparisons to be made.
D. Synthesis of Acetylcholine
The synthesis of ACh from Ch and acetyl coenzyme A requires the enzyme choline acetyltransferase (ChAc); the acetylation of coenzyme A
(CoA) occurs through the action of ATP and requires a source of active acetate.
a) Coenzyme A and active acetate. Coenzyme A is a ubiquitous
component of nerve cells and its supply is not limiting in ACh synthesis
except, perhaps, during pantothenic acid deficiency (Novelli, 1953). 15
Because of its active involvement in numerous metabo1ic cycles, CoA can be derived from protein, carbohydrate and fat dietary sources. Its absence in circulating plasma suggested that each CoA containing tissue can synthesize its own supp1y as needed (Novel li , 1953).
Active acetate is also readily obtained from a variety of sources
(Novelli, 1953). Glucose was necessary for ACh synthesis in brain slices or in perfused superior cervical ganglia; lactose or pyruvate substituted for glucose, but acetate, acetoacetate, or succinate could not (Quaste1,
Tennenbaum and Wheat1ey, 1936; Kahlson and MacIntosh, 1939; Browning and
Schulman, 1968).
b) Choline. The stimulated superior cervical ganglion released more
ACh than it initially contained even when it was perfused with a Ch-free solution (Brown and Feldberg, 1936b; Kahlson and MacIntosh, 1939; Perry, 1953;
Birks and MacIntosh, 1961; Matthews, 1963). Therefore some ACh synthesis occurred in the absence of externa11y added Ch and it is still not entirely c1ear where the Ch for this synthesis came from. The presynaptic pool of Ch in ganglia was shown by Friesen, Ling and Nagai (1967) to be about 50 ng, and it was therefore too sma11 ta support ACh synthesis for more than a few min of stimulation. The large amounts of Ch that appeared in the effluent
from resting gang1ia perfused with Ch-free solution may have provided some
of the Ch necessary for ACh synthesis, but the origin of this Ch is not known. Nervous tissue contains large amounts of bound Ch in phospho1ipid
and it is possible that some of this was avai1ab1e for ACh synthesis.
The turnover of phospho1ipid-Ch, as measured by Collier and Lang (1969),
wau1d release only 2 ng/min and if this was the source of Ch for ACh
synthesis ,in gang1ia perfused without Ch a11 of it wou1d have to be 16
available for ACh synthesis. Perhaps the ganglion which lacks exogenous
Ch can break down bound Ch more rapidly than can a normal preparation.
It is not-clear why the Ch that was available for ACh synthesis in a stimulated ganglion seemed to be unavailable for the synthesis of surplus
ACh in a resting ganglion; an eserinized ganglion perfused without Ch made about 60 ng of surplus ACh in the first few min, and this was probably from the endogenous Ch, but no more surplus ACh was synthesized when perfusion was continued (Birks and MacIntosh, 1961).
Despite the ganglion's ability to synthesize some ACh in the absence of added Ch, the preparation must be provided with a source of extracellular
.Ch for optimal synthesis and release. Brown and Feldberg (1936b) perfused ganglia with Ch-free Locke and showed that when ACh release from the stimulated ganglion began to decrease, the addition of Ch to the perfusion fluid enhanced both the ACh output and the nictitating membrane contraction.
Birks and MacIntosh (1961) confirmed that the output of ACh from ganglia was maintained at a higher level when perfusion was with eserine-Ch-Locke than it was when perfusion was with es erine-Ch free -Locke. In the presence of
Ch, synthesis of ACh was able to match its release, and a ganglion's ACh
content turned over about 4 times during 1 hr's stimulation (Birks and
MacIntosh, 1961; Matthews, 1963). Cat's plasma contains about 10-5 M
free choline and this level is maintained remarkably constant (Bligh, 1952);
it is not likely that Ch becomes deficient under physiological conditions.
A way of limiting the supply of Ch for ACh synthesis became available
with the synthesis of HC-3 (Schueler, 1955). This substance inhibited ACh
synthesis by intact cells (see e.g. MacIntosh et al., 1956; Gardiner, 1961;
Birks and MacIntosh, 1961) in concentrations that had no inhibitory effect 17
on ChAc (MacIntosh et al., 1956; Gardiner, 1957; Gardiner, 1961); HC-3 probably competed with Ch for carrier sites (MacIntosh et al., 1956;
Gardiner, 1957; MacIntosh, 1961). In the presence of HC-3, ACh release from the stimulated superior cervical ganglion was not maintained, and the ganglion's content of releasable transmitter was depleted. The addition of Ch antagonized the action of HC-3 (Birks and MacIntosh, 1961;
Matthews, 1966). HC-3 also prevented the formation of surplus ACh by starving the ganglion of the Ch necessary for the synthesis of surplus ACh
(Birks and MacIntosh, 1961).
Although plasma is an important source of Ch for ACh synthesis, Ch
formed from the hydrolysis of released ACh can be re-used by the ganglion.
This was first suggested by Perry (1953) from experiments w~ich showed that
the evoked release of ACh in the presence of eserine was greater than the
release of Ch in the absence of eserine. Perry's suggestion was confirmed
by Collier and MacIntosh (1969) who showed that ganglia which had been
loaded with 3H- ACh released twice as much labelled material upon pre
ganglionic nerve stimulation when eserine was present than when eserine was
absent. These experiments were interpreted as evidence that Ch formed
from the hydrolysis of ACh in the absence of eserine can effectively compete
with plasma Ch for transport into nerve endings; the alternative explanation
that eserine increased the release of transmitter cannot be entirely
eliminated. The re-uptake of ACh itself was disregarded by these authors
because ACh in the presence of eserine was not accumulated by ganglia.
However, they did not examine ACh uptake in eserine's absence, and it is
now known that this anti-ChE prevents ACh accumulation by brain tissue
(Polak and Meeuws, 1966; Schuberth and Sundwall, 1967; Polak, 1969; Liang and 18
Quaste1, 1969); ACh uptake into brain tissue can on1y be demonstrated if an organophosphorus an ti-ChE is used instead of eserine.
The manner in which Ch gains access into nerve endings is not yet entire1y c1ear. Carrier uptake against a concentration gradient of Ch has been described in a variety of tissues: into kidney slices (Sung and Johnstone, 1965), erthrocytes (Askari, 1966; Martin, 1967, 1968), squid giant axons (Hodgkin and Martin, 1965), and brain slices (Schuberth,
Sundwa11, Sorbo and Lindell, 1966; Schuberth, Sundwa11 and Sorbo, 1967); faci1itated diffusion has been described for Ch uptake into synaptosomes
~rchbanks, 1968b; Potter, 1968; Diamond and Kennedy, 1969). Whether transport of Ch into these tissues was against the e1ectrochemica1 gradient is not known. The active uptake of Ch into brain slices (Schuberth et al.,
1967) or red ce11s (Martin, 1967, 1968) has been shown to be Na+ dependent, but the effects of Na+ on the faci1itated transport of Ch into synaptosomes is contradictory (Marchbanks, 1968~; Potter, 1968; Diamond and Kennedy, 1969).
The synthesis of ACh by superior cervical gang1ia (Birks, 1963) or by brain
(Bhatnagar and MacIntosh, 1967) was reduced in low Na + medium and this may be the result of decreased Ch transport.
c) Choline acetyltransferase and the subcéllu1ar location of acety1cho1ine synthesis. ChAc was first iso1ated by Nachmansohn and Machado
(1943) from extracts of brain and electric organ of the ee1 and was later iso1ated from the mamma1ian nervous system (Nachmansohn and John, 1944, 1945).
The enzyme transfers acety1 groups from acety1 CoA to Ch and although it is fair1y specific it can acety1ate substances structura11y similar to Ch
(Burgen, Burke and Desbarats-Schonbaum, 1956) and it can synthesize Ch esters other than ACh (Berman, Wilson and Nachmansohn, 1953; Berry and Whittaker, 19
1959). ChAc is present in cholinergic neurons (Cohen, 1956; Hebb and
Silver, 1956) and it appears to be synthesized in the cell body and transported to the periphery by axoplasmic flow (Hebb and Waites, 1956).
Hebb and Waites (1956) demonstrated that decentralized sheep or cat superior cervical ganglia lost much of their ChAc;only about 10% of the enzyme remained 1 week after the nerve was cut. Decentralization a1so resulted in the parallel loss of the ganglion's ACh (MacIntosh, 1938;
Feldberg, 1943; Banister and Scrase, 1950; Friesen et al., 1967).
'The exact location of ChAc within the nerve ending is not yet entirely clear. Present evidence from subcellular fractionation studies suggests that it is a soluble cytoplasmic enzyme, although earlier studies were equivocal. First attempts to employ the techniques of differential centrifugation to brain showed that approximately 60% of the tissue's ChAc activity was associated with mitochondria (Hebb and Smallman, 1956), but with more refined techniques it was shown that this was not true (Hebb and
Whittaker, 1958).
De Robertis et al. (1963) isolated synaptosomes from rat or rabbit brain homogenates and showed the association of both ACh and ChAc with synaptic vesicles; they suggested that vesicles are both the site of storage and synthesis of ACh. However, Whittaker et al. (1964) who used homogenates of pigeon or guinea-pig brain demonstrated that ChAc is a soluble cyto- plasmic enzyme. These different results were due partly to a species difference and partly to the different techniques used in isolating the subcellular fractions CMcCaman, Rodriguez de Lores Arnaiz and De Robertis,
1965). Fonnum (1966, 1967) showed that the procedures used by Whittaker
and those used by De Robertis favoured the results they obtained in their 20
respective species. Many low molecular weight pro teins are known to be relatively insoluble in media of low ionic strength, and they therefore appear to be membrane bound, but they are soluble in the presence of electrolytes (see e.g. Schneider, 1963). Fonnum showed this to be true for the rat brain ChAco At the pH and ionic strength likely to be found in the intact nerve-ending, 80% of the ChAc appeared free in the cytoplasm but at the low ionic strengths used in earlier experiments the enzyme could be adsorbed onto membranes; this adsorption of ChAc varied with species and was low in those used by Whittaker et al. and high in those used by
De Robertis et al. Potter, Glover and Saelens (1968) have confirmed
Fonnum's findings on rat brain ChAco
The evidence from subcellular fractionation studies of ChAc and ACh has led to the suggestion that ACh synthesis takes place in the cytoplasm and the transmitter is then packaged into vesicles (Beani et al., 1969).
Unless the process is more complicated than this, this suggestion was not supported by the present experiments that measured the mixing of surplus ACh
(probably cytoplasmic) with ACh that is releasable by nerve impulses
(probably vesicular).
E. Acetylcholinesterase and the Fate of ReleasedAcetylèholine
In 1914, Dale demonstrated the transient action of injected ACh and
suggested that the ester was quickly destroyed by an enzyme. The existence
of such an enzyme has since been demonstrated in a wide variety of animal
tissues, including nervous tissues of vertebrates and invertebrates: e.g.,
the electric organ of Electrophorus Electricus (Rothenberg and Nachmansohn,
1947), squid giant axon (Nachmansohn and Rothenberg, 1945), mammalian brain 21
(Nachmansohn and Rothenberg, 1945), superior cervical ganglion of the cat (v. Brucke, 1937; Glick, 1937), and the neuromuscular junction
(Koelle and Friedenwald, 1949).
Eserine inhibited the action of cholinesterases (ChE) and this test distinguished ChE from other non-specifie esterases (Richter and
Croft, 1942). It is now apparent that there are at least two types of
ChE enzymes (see e.g. Augustinsson, 1963); acetylcholinesterase (AChE), whose function is to destroy ACh, rapidly hydrolyses ACh; pseudocholin
esterases (as butyrocholinesterase), whose physiological function is
unknown, also hydrolyses ACh, but at a slower rate than do es AChE
(Augustinsson and Nachmansohn, 1949). Although both "true" and "pseudo"
ChE are inhibited by eserine, they can be differentiated by other
pharmacological properties (see reviews by Holmstedt, 1959, and
Augustinsson, 1963).
a) Neuronal barriers to cholinesterase inhibitors. Schweitzer and
Wright (1937a, 1937b), and Schweitzer, Stedman and Wright (1939) showed that
the quaternary ammonium anti-ChE agent, neostigmine, penetrated the blood
brain barrier less readily than did the tertiary amine, eserine. This
was confirmed by Burgen and Chipman (1952) who correlated the lipid
solubility (as olive oil/water distribution coefficients) of various anti
ChE agents with the inhibitior. of brain AChE following their parenteral ad-
ministration. Tetraethylpyrophosphate (TEPP) and eserine had about equal
lipid solubility, diisopropy1fluorophosphate (DFP) was more soluble,
neostigmine had a partition coeffecient 100 times sma1ler than TEPP or
eserine, and phosphopyristigmine (pPS) was almost insoluble in the olive
oi1; of these anti-ChE agents only TEPP, DFP and eserine affected the 22 e central nervous system. Similar results were obtained by Koelle and Steiner (1956) who used two potent thiophosphate anti-ChE agents: a
tertiary amine, 2-diethoxyphosphinylthioethyldimethylamine acid oxalate
(2l7-AO), and a quaternary ammonium agent, 2-diethoxyphosphinylthioethyl
trimethylammonium iodide (2l7-MI).
The relative permeability of squid giant axon to quaternary and
tertiary anti-ChE agents was studied by Bullock, Nachmansohn and
Rothenberg (1946) and Feld, Grundfest, Nachmansohn and Rothenberg (1948);
eserine and DFP penetrated the axonal membrane, but neostigmine did not.
Nachmansohn (1950) attributed these differences to the chemical structure
of these compounds and suggested that the axonal membrane may be an
effective barrier to quaternary ammonium substances (including ACh).
b) Functional and residual AChE. Koelle (1951) used histochemical
techniques to study the distribution of AChE in superior cervical ganglia;
he showed that most of this enzyme is located in presynaptic terminals
because chronically decentralized ganglia did not stain for AChE. Koelle
(1957) and Koelle and Koelle (1959) showed that part of this AChE is
intracellular, and part of it is extracellular. In those experiments,
cats were injected with a lethal dose of DFP, and this was preceded in some
animals by sublethal doses of ambenonium, a bis-quaternary anti-ChE which
does not penetrate cell membranes; some animals were not treated with any
anti-GhE drug. The superior cervical ganglia of these cats were removed
and stained for AChE: ganglia from untreated animals were heavily stained;
ganglia from cats treated with only DFP did not stain, but ganglia whose
external AChE had been protected from DFP by pretreatment with ambenonium
stained only on external surfaces (ambenonium protected the external AChE 23
from DFP in the whole animal, but could be removed since it is a reversible anti-ChE agent). Koelle and Koelle (1959) suggested tbat internaI (or "residual") AChE is synthesized within the endoplasmic reticulum at the perikaryon and is then transported within the axon to its external (or. "functional") sites.
It is now clear that the external AChE is responsible for breaking down released ACh. Phosphopyristigmine which is lipid insoluble and therefore does not enter nerve cells, and eserine, which is lipid so1uble and thus easily penetrates nerve cells, were equally effective in pre serving the ACh released from the stimulated superior cervical ganglion of the cat (Burgen and Chipman, 1952). Similar results were obtained by
Emmelin and MacIntosh (1956) who compared ACh release from ganglia perfused with neostigmine, eserine, TEPP, or DFP; release was about the same which ever agent was used. The same conclusion was reached by Mc Isaac and Koelle
(1959) who showed that ganglionic potentials and nictitating membrane contractions, induced by preganglionic nerve stimulation of the partially resected superior cervical sympathetic nerve,were potentiated equally by the tertiary (2l7-AO) or by the quaternary (2l7-MI) anti-ChE agent.
The accumulation of surplus ACh by a superior cervical ganglion was described in section C above. The most likely explanation of its formation is that the synthesis of ACh is continually occurring in tbe resting ganglion, probably ~ithin the nerve ending cytoplasm; in the absence of a "vesicular need", this newly-synthesized transmitter is destroyed by the cytoplasmic or residual pool of AChE. The inhibition of intracellular AChE by eserine allows the accumulation of surplus ACh.
If this explanation of the formation of surplus ACh is correct, it would 24
be expected that surplus ACh would accumulate less readily in the presence of a lipid insoluble anti-ChE than it would in the presence of lipid soluble eserine. This was tested in the present work by comparing the formation of surplus ACh in eserine medium with its formation in neostigmine or ambenonium solution.
c) The physiological role of. AChE in the superior cervical ganglion.
Various roles have been postulated for AChE in synaptic transmission in the superior cervical ganglion of the cat. Feldberg and Vartiainen (1934) and Eccles (1944) postulated that the function of AChE is to limit trans mitter action at postsynaptic fibres, but if most of the enzyme is located presynaptically (Koelle and Koelle, 1959), it is poorly placed for this function. Emmelin and MacIntosh (1956) suggested diffusion from the synaptic cleft (see also Ogston, 1955) and ultimate destruction by blood
ChE may be th~ most important way in which the transmitter's action is terminated. Koelle and Koelle (1959) suggested that the enzyme, located presynaptically, might protect presynaptic fibres from endogenously r~leased
ACh. Later, Koelle (1961, 1962) hypothesized that the ACh released by nerve action potentials from presynaptic terminaIs is too little to
depolarize postsynaptic elements, but might induce additional release of
ACh; AChE would prevent an exaggerated release. The suggestion that ACh
releases ACh was supported by the findings of VoIle and Koelle (1961) who
found that a chronically decentralized superior cervical ganglion was less
sensitive to injected carbachol or, in the presence of DFP, to ACh than
was a normally innervated preparation. These experiments of VoIle and
Koelle were interpreted as evidence that carbachol and ACh release
endogenous ACh from the presynaptic fibres, which have a greater sensitivity 25
to these drugs than have postsynaptic e1ements. Although simi1ar experiments with carbacho1 have fai1ed to show any difference between normal and decentra1ized gang1ia (Brimb1ecombe and Sutton, 1968; Brown,
1969), McKinstry, Koenig, Koe1le and Koelle (1963) and McKinstry and
Koe1le (1967) demonstrated clearly thatcarbachol does release ACh from the cat superior cervical ganglion. In these experiments McKinstry and her colleagues injected carbachol into ganglia that were perfused with eserine-Ch-Locke and they measured by bio-assay the vasodepressor activity of the effluent; after placing the samp1es in a boiling water bath, which destroyed the ACh but not the carbacho1, each samp1e was re-assayed and the difference between the two results gave a measure of the amount of ACh released by the injected carbacho1. Because these authors perfused with solution containing Ch and eserine, the ganglia would synthesize and store surplus ACh (Birks and MacIntosh, 1961). It was not c1ear how much of the carbachol induced ACh release came from the surplus pool and how much came from the transmitter (depot) pool, nor cou1d McKinstry et al. test
ACh itse1f. In the present work, a method was devised whereby ACh or carbachol could be tested directly for an effect upon surplus ACh release. 26
II. METHODS AND MATERIALS 27
A. Ganglion Perfusion
Adult cats (2-3 kg) of either sex were used; in most experiments anaesthesia was induced by ethyl chloride followed by ether and maintained by chloralose (i.v., 80 mg/kg), but in a few experiments sodium pentobarbital (i.p., 30 mg/kg) was used. If necessary, small doses of sodium pentobarbital (3-10 mg) were given i.v. during the experiment to main tain anaesthesia. After cannulating the trachea, the right superior cervical ganglion was prepared for perfusion by Kibjakow's (1933) proce dure as described by Feldberg and Gaddum (1934). The transverse vein was cut between double ties, the cervical lymph node was removed, and aIl branches of the common carotid artery, except those supplying the ganglion were tied off. The external carotid artery, the lingual artery, and if present the internaI carotid artery, were aIl tied and cut, and the occipital artery was prepared for ligation. If the cat had an internaI jugular vein, aIl its branches were tied except the one that drained the ganglion; in the absence of an internaI jugular vein, the transverse prevertebral vein was prepared for cannulation. Arterial twigs at the
level of the postganglionic fibres were tied carefully, and a thread was placed around the vagus nerve rostral to the nodose ganglion. The
occipital artery was tied immediately before the start of perfusion, and
the vagus nerve above the nodose ganglion, and internaI jugular vein (or
transverse prevertebral vein) were tied just after the start of perfusion.
Thus the ganglion was completely isolated from its blood supply.
Perfusate was introduced through a small glass cannula that was inserted
high in the commcn carotid artery; the effluent was collected in chilled
test tubes from the internaI jugular vein or transverse prevertebral 28
vein, and either assayed immediate1y or stored at -15°C for no longer than 24 hr before assay.
Perfusion was with Locke solution (composition in g/l: NaC1
8.2, KC1 0.42, CaC1 0.24, glucose 1.0, and NaHC0 1.35), which contained 2 3 one or more of the fo11owing: choline ch10ride (Ch, 1.5 ~g/m1), either 3 un1abe11ed or methy1- H 1abe11ed (63 mc/m-mo1e), eserine su1phate
(10 ~g/m1), neostigmine bromide (10 ~g/m1), ambenonium ch10ride (10 ~g/m1), diisopropy1f1uorophosphate (DFP, 10 ~g/m1), hemicho1inium #3 dibromide
(HC-3, 10 ~g/m1), acety1cho1ine ch10ride (ACh, 0.5-15 ~g/m1), carbacho1
(1-30 ~g/m1). The concentration of Ch used was close to the physio1ogica1
1eve1 of Ch in cat plasma (B1igh, 1952). When perfusing with solutions containing high K+ , the KC1 concentration of Locke was raised 10 times to 4.2 g/l (56 mM) and NaC1 was reduced by an iso-osmotic amount.
The Locke solution was fi1tered before use and was equilibrated with
3.5% CO in 02 throughout the experiment to maintain a pH of approximate1y 2 7.4 at body temperature. Each solution was p1aced in a separate 250 ml conica1 bott1e which was connected through narrow polyethylene tubing to a 22 gauge need1e. The need1es carrying solution from the bott1es were pushed through a rubber stopper into the glass cannu1a. Perfusion cou1d be switched from one solution to another by turning the appropriate stopcock, the dead space being on1y the volume of the arterial cannula.
The tubes carrying solution from the reservoir to the cannu1a were passed up the oesophagus from its point of exit be10w the diaphragm to an exit just be10w the 1evel of the ganglion so that the perfusion fluid was warmed to body temperature (Emmelin and MacIntosh, 1956). Perfusion pressure was contro1led by connecting the perfusion bott1es through a 29
reservoir (20 litres) to a cylinder containi~g 3.5% CO2 in O2 , and the perfusion rate maintained at approximately 0.3 ml/min by adjusting the perfusion pressure over the range of 40-100 mm Hg. Drugs were injected directly into the arterial cannula in 0.2 ml Locke solution.
B. Experimental Procedure
In all experiments, perfusion for the first 15 min was with Ch-
Locke. This allowed time for the unhurried cannulation of the vein, for adjustments of perfusion pressure, and for a short test stimulation of the preganglionic nerve. After this initial 15 min perfusion, one of the following procedures was used.
a) Formation of surplus acetylcholine. In experiments that tested the rate of formation of surplus ACh, the ganglion was perfused at rest
(no stimulation) for 15-150 min with Ch-Locke containing either eserine, neostigmine or ambenonium. At the end of this time the ganglion was removed for bio-assay and its ACh content was compared to the control unperfused ganglion.
b) Labelling of surplus acetylcholine. In experiments which were
designed to label surplus ACh, the initial 15 min perfusion was followed
by perfusion for 60 min with Locke containing 3H- Ch in the presence of
either eserine or DFP; in some experiments the ganglion was removed at
the end of this loading perfusion. In other experiments, the loading
perfusion was followed by a washout perfusion with unlabelled Ch-eserine
(or DFP)-Locke and various procedures were tested for their ability to
release the labelled surplus ACh. In the experiments designed to test
release of surplus ACh by injected or perfused drugs it was necessary 30
to flush the tubing through which the test solution was to be applied because a small amount of radioactive solution diffused into its tip during the perfusion with labelled Ch. Half a ml of Locke solution was injected through the tubing just before the test to make sure that no artefact would be measured. When high K+ was tested, the perfusion pressure was increased by about 30% to compensate for the vasoconstriction caused by high K+ •
c) Release of acetylcholine into different anticholinesterase- containing Locke solution. Experiments were designed to measure ACh release into Ch-Locke solution containing one of three anti-ChE agents
(eserine, neostigmine, or ambenonium). After the initial 15 min perfusion with Ch-Locke, the perfusion medium was switched to one containing Ch and an anti-ChE drug; ACh release was tested 5 min later. The test was preganglionic nerve stimulation (20/sec, 0.8 msec, 5 V) for 4 min and successive tests were separated by 10 min perfusion (no stimulation) with
Ch-Locke. In each experiment aIl three anti-ChE agents were tested separately, but the or der of exposure to the compounds was changed from one experiment to the next.
C. Chronic Decentralization of Superior Cervical Ganglia
Cats were anaesthetized with sodium pentobarbital (i.p., 30 mg/kg) and the skin of the neck.was washed with 2% KI, 2% l in 70% ethanol.
AlI surgical instruments were sterilized in boiling water and a 70% ethanol solution. A mid line incision was made and both superior cervical sympathetic nerves were exposed carefully low in the neck. The pre- ganglionic nerves were cut and a few mm of nerve removed; the wound was 31
closed with sterile surgical thread. All animals were given 200,000 i.u. of penicillin G pro caine and 0.25 g streptomycin sulphate (Fortimycin-! suspension) as an i.m. injection immediately after the operation. The animals were allowed to recover and were used for perfusion one week after the operation.
D. Stimulation of the Preganglionic Sympathetic Nerve
The super{or cervical sympathetic nerve was separated carefully from the vagus nerve and from connective tissue, and cut low in the neck whether it was to be stimulated or not. The nerve was placed on platinum wire electrodes, bathed in warm paraffin oil, and stimulated when necessary with supramaximal rectangular pulses (5-10 V, 0.8 msec, 3-20/sec).
When the nerve was stimulated for long periods of time, the electrode was moved a few mm proximally along the nerve every 5 min. A thread was tied to the nictitating membrane and attached to a Grass force transducer; isometric contractions of the membrane were monitored on a Gilson or Grass pen recorder.
E. Ganglion Extracts
The control unperfused ganglion was removed before starting to perfuse the test ganglion. Perfusion of the test ganglion was continued mltil its removal; at the end of the experiment, the ganglion was removed quickly, dipped briefly in 0.9% NaCl and the tissue was minced finely in
2.0 ml ice cold 10% trichloroacetic acid (TCA) solution. This procedure
extracts all ACh (Chang and Gaddum, 1933).
The tissue suspension was a110wed to stand at O°C for 90 min; the 32
supernatant was decanted, the residue was washed with 1.0 ml of TCA
solution, and the wash combined with the original supernatant. The
TCA was removed by shaking the extract 5 times with 5 volumes of water
saturated ether, the solutions were then aerated briefly to remove any
trace of ether and, when necessary, the volume was made up to 3.0 ml with 0.9% NaCl. In all experiments, a blank containing only 10% TCA was carried through the same procedure. An aliquot of the blank
solution rarely produced a response in the bio-assay for ACh; if it did,
blank and test solutions were aerated for an additional few min to remove
any ether still remaining.
Aliquots of the final extract were used for bio-assay, for liquid
scintillation counting, or for the separation of labelled ACh from other
labelled material.
F. Bio-assay
The ACh content of ganglion extractsor effluents was estimated
by bio-assay on the blood pressure of the eviscerated cat (MacIntosh and
Perry, 1950). Young cats (l.5-2.5 kg) were anaesthetized with intravenous
chloralose (80 mg/kg), the trachea was cannulated, and a mid line abdominal
incision was made. In the following order: the rectum, the inferior and
superior mesenteric arteries, the colic artery, the oesophagus and the
portal vein were cut between double ties; the gastrointestinal tract
together with the spleen was then removed by cutting connective fascia.
The abdomen was closed after checking for bleeding and moistening internal
structures with 0.9% NaCl.
Twenty ml of 0.9% NaCl was then injected slowly into the femoral 33
veine The carotid artery was cannu1ated and connected to a Statham pressure transducer by polyethylene tubing fi11ed with heparinized saline. B100d pressure was recorded on a pen recorder (either Gilson or Grass pen po1ygraph). When necessary, b100d pressure was maintained at approximate1y 100-120 mm Hg by the intermittent infusion of nor adrena1ine (1 ~g/m1) through the arteria1 cannu1a (Quaste1, 1962). The reservoir containing noradrena1ine had a pressure head of 100-120 mm Hg, and noradrena1ine was therefore on1y infused when the b100a pressure fe11 be10w this 1eve1. If breathing was irregu1ar and resu1ted in b100d pressure fluctuations, the vagus nerves were cut and the cat was artificia11y respired.
The animal was tested for sensitivity to ACh by injecting the drug i.v.; if it did not respond to 3 ng, eserine su1phate (0.1 mg/kg) was injected to increase the sensitivity to ACh. ACh (as the ch10ride) was di1uted in a solution of simi1ar composition as the materia1 to be assayed, and its concentration adjusted to approximate that of the assay materia1 so that simi1ar volumes of both cou1d be injected. Doses of the test samp1e and known doses of ACh were a1ways injected a1ternate1y at 2 min interva1s and were washed into the animal with 1.5 ml of 0.9% NaC1.
The concentration of the test samp1e was estimated by comparing its
depressor effects to those produced by known amounts of ACh, and an attempt was a1ways made to assay at more than one dose 1eve1. A11 values of ACh
are given as their ch1orides.
The depressor effects of both the test samp1e and authentic ACh were
a1ways abo1ished by treating the samp1e with a1ka1i, or by pretreating the
cat with atropine (0.1 mg/kg). A typica1 assay is shown in Fig. 1; in 34 e
l ' t t t 3ngACh Sng ACh 10ngACh 0.1 ml B
t t t 1 10 ng ACh 0.05 ml , 5 n9 ACh
FIG. 1. A typica1 bio-assay for the estimation of ACh extracted from a catIs superior cervical ganglion. "B" rep,":ç.sents a 0.1 ml injection of a control solution (see text); "T" represents a test injection of a ganglion extract. The ACh concentration of the test samp1e was estimated to be 100 ng/m1. 35
this cat, 3, 5 and 10 ng of ACh were readily differentiated, 0.1 ml of the blank gave no depressor response, but the response of 0.1 ml of the test solution was close to that produced by 10 ng ACh and
0~05 ml of the test produced an effect similar to that of 5 ng ACh.
Thus, the test sample was estimated to contain 100 ng ACh/ml.
G. Separation of Labelled Material
Labelled ACh was separated from other radioactive material in ganglion extracts or effluents by the procedures described by Collier and Lang (1969); the methods were based upon those of Shaw (1938), and
Saelens and Stoll (1965) for the reineckate precipitation test and of
Kapfhammer and Bischoff (1930) and Dudley (1933) for the gold pre- cipitation test.
One ml 3H- ACh solution (50 ng), 1.0 ml 3H- Ch solution (50 ng),
1.0 ml of ganglion extract or 1.0 ml of ganglion effluent was placed into separate test tubes and these were used for either the selective pre- cipitation by goldor by reineckate.
a) Separation by go Id chloride. Five mg carrier ACh (in 0.2 ml water) and 0.4 ml of a 10% gold chloride solution were added to each test tube. The precipitate was allowed to form for 60 min at room temperature after which it was collected by centrifugation. Radioactivity in aliquots of the supernatant was determined; the precipitate was dissolved in acetone and measured for radioactivity.
b) Separation byreineckate. To each test tube containing either 1.0 ml
3H- ACh solution (50 ng), 1.0 ml 3H- Ch solution (50 ng), 1.0 ml of ganglion
extract or 1.0 ml of ganglion effluent, 1.0 mg carrier Ch and 1.0 mg 35
this cat, 3, 5 and 10 ng of ACh were readily differentiated, 0.1 ml of the blank gave no depressor response, but the response of 0.1 ml of the test solution was close to that produced by 10 ng ACh and
~5ml of the test produced an effect similar to that of 5 ng.ACh.
Thus, the test sample was estimated to contain 100 ng ACh/ml.
G. Separation of Labelled Material
Labelled ACh was separated from other radioactive material in
ganglion extracts or effluents by the procedures described by Collier
and Lang (1969); the methods were based upon those of Shaw (1938), and
Saelens and Stoll (1965) for the reineckate precipitation test and of
Kapfhammer and Bischoff (1930) and Dudley (1933) for the gold pre-
cipitation test.
One ml 3 H-ACh solution (50 ng), 1.0 ml 3 H-Ch solution (50 ng),
1.0 ml of ganglion extract or 1.0 ml of ganglion effluent was placed into
separate test tubes and these were used for either the selective pre-
cipitation by gold 'or by reineckate.
a) Separation by gold chloride. Five mg carrier ACh (in 0.2 ml
water) and 0.4 ml of a 10% gold chloride solutionwere added to each test
tube. The precipitate was allowed to form for 60 min at room temperature
after which it was collected by centrifugation. Radioactivity in aliquots
of the supejrnatant was determined; the precipitate was dissolved in acetone
and measured for radioactivity.
b) Separation by reineckate. To each test tube containing either 1.0 ml
3H- ACh solution (50 ng), 1.0 ml 3H-Ch solution (50 ng), 1.0 ml of ganglion
extract or 1.0 ml of ganglion effluent, 1.0 mg carrier Ch and 1.0 mg 36
carrier ACh were added (in 0.1 ml water). Five ml of a saturated aqueous solution of ammonium reineckate was added to aIl test tubes and the precipitate allowed to form for 60 min at ~oom temperature.
The precipitate was collected by centrifugation, the supernatant was discarded, and the precipitate was shaken with a 7:1 v/v mixture of
40% ethanol and saturated reineckate solution. The ethanolic suspension was warmed to 57°C for 3 min and was then allowed to stand at room temperature for 30 min. The final precipitate was collected by centrif- ugation; radioactivity in aliquots of the supernatant was measured and the precipitates were dissolved in acetone for liquid scintillation counting.
The proportion of labelled ACh in the test samples was calculated algebraically from the partition of authentic 3 H-ACh and 3 H-Ch. The behavior of standard ACh or Ch was determined each time the tests were
used. The approximate partition of radioactive Ch and ACh between the
final supernatant and precipitate in the two methods used was as follows:
i) for gold: ACh - 27% in the supernatant and 73% in the precipitate;
Ch - 84% in the supernatant and 16% in the precipitate. ii) for reineckate:
ACh - 36% in the final supernatant and 64% in the precipitate; Ch - 6% in
the final supernatant and 94% in the final precipitate.
Labelled phosphorylcholine was present in extracts of ganglia that
had been perfused with radioactive Ch; this material was not precipitated
by initial reineckate precipitation and is mostly discarded with the first
supernatant in that test. However, in the gold test phosphorylcholine
remained in the supernatant along with unchanged Ch. The difference
between the total radioactivity in the unknown sample and the total ' 37
radioactivity precipitated by ammonium reineckate gave the amount of phosphorylcholine in the sample; correction for the contribution of
phosphorylcholine to radioactivity in the supernatant of the gold test
could then be made. After correcting for phosphorylcholin~ results from
the gold and reineckate separations generally agreed within 10-15%.
H. Acetylation of 3 H-Choline
The 3H- ACh that was used in the selective precipitation tests was
prepared from 3H- Ch by the procedure described by Emmelin and MacIntosh
(1956). Five hundred ng of 3 H-Ch in aqueous solution was evaporated to
dryness in an ampoule, which was then cooled in ice, and 1.0 ml of ice
cold acetyl chloride was added; the ampoule was then sealed and placed in
boiling water for 15 min. After cooling, the ampoule was opened, excess
acetyl chloride was removed with the aid of a stream of air, and the 3H- ACh
was dissolved in distilled water.
I. Measurements of Radioactivity
Radioactivity in aliquots of ganglion extracts or effluents was
determined by liquid scintillation spectrometry (either Picker Nuclear,
Liquimat-330, or Nuclear Chicago, Mark 1); a minimum of 3500 counts was
accumulated 50 that the counting error was always 5% or less. The solvent
system consisted of: l litre toluene, 600 ml ethylene glycol monomethyl
ether, 6.4 g 2,5-diphenyloxazole (PPO), 80 mg p-bis [2-(5-phenyloxazolyl)]-
benzene (POPOP). 3 H activity of a 0.2 ml aliquot of ganglion effluent
in 15 ml of this mixture could be counted with 20-25% efficiency; this
mixture would incorporate up to 4% water. Quench correction was made by 38
the external standard channels-ratio method, and was calculated by hand from curves or by a programmed PDP-8 computer (Digital Equipment
Corporation). In the above solution, the efficiency of counting radio- activity in coloured samples from the selective precipitation tests was low due to heavy quenching; these samples were therefore counted,in
a solvent system containing 1 litre toluene, 1 litre ethanol, 1 litre
dioxane, 240 g napthalene, 15 g PPO, and 187 .5mg POPOP. This solvent system incorporated up to 10% water; with 0.2 ml of water in 15 ml of
solvent the efficiency was 20-25% and with 0.5 ml of reineckate solution
in 15 ml of solvent the efficiency was about 15%.
J. Materials
The following drugs and chemicals were used in these experiments:
Ethe~ (E.R. Squibb and Sons Ltd.)
Ethyl chloride (J.F. Hartz Co. Ltd.)
Chloralose Ckindly provided byHoffmann-LaRoche Ltd.)
Sodium pentobarbital (Nembutal, Abbott Laboratories Ltd.)
Eserine sulphate (Nutritional Biochemicals Corp.)
Neostigmine bromide (Sigma Chemical Co.)
Ambenonium chloride CMytelase, kindly provided by Dr. C.W. Berkett.)
Diisopropylfluorophosphate (DFP, K and K Laboratories.)
Choline chloride (Ch, British Drug Houses Ltd.)
3H-Choline chloride (3H- Ch , 63 mc/m-mole, New England Nuclear.)
Acetylcholine chloride (ACh, British Drug Houses Ltd.)
Carbachol (K and K Laboratories.)
Atropine sulphate (British Drug Houses Ltd.) 39
Hemicholinium #3 dibromide (HC-3, kindly provided by Dr. V.B. Haarstad.)
Acetyl chloride (J.T. Baker Chemical Co.)
Noradrenaline hydrochloride (Nutritional Biochemicals Corp.)
Penicillin G Procaine in aqueous suspension with streptomycin sulphate
(Fortimycin-! suspension, Ayerst Laboratories.)
Trichloroacetic acid (TCA, J.T. Baker Chemical Co.)
Heparin sodium (Nutritional Biochemicals Corp.)
Ammonium reineckate (J.T. Baker Chemical Co.) Gold chloride (J.T. Baker Chemical Co.)
2,S-Diphenyloxazole (PPO, New England Nuclear.) p-bis[2-(S-Phenyloxazolyl)]-benzene (POPOP, New England Nuclear.)
All other chemicals used were Baker Analysed Quality.
K. Statistical Analysis
In the statistical evaluation of the results, Students lit" for ungrouped data was l.Jsed as a test ofsignificance. Two-tailed significance tests were used throughout. The standard deviation (S.D.) was used as the measure of deviation. 40
III. RESULTS 41
A. Formation of Surplus Acetylcholine in. the Presence of Various
Anticholinesterase Agents
These experiments measured the rate of .surplus ACh formation by perfused resting superior cervical ganglia. The results (Fig. 2) showed
that accumulation of surplus ACh was delayed when neostigmine or ambenonium was used instead of eserine.
Resting ganglia were perfused with Ch-Locke for 15 min, and then with
eserine-Ch-Locke (4 experiments), or with neostigmine-Ch-Locke (21 experi ments) or with ambenonium-Ch-Locke (2 experiments) for 20-150 min; at the
end of this time the perfused ganglia were removed, extracted with TCA, and
their ACh content determined by bio-assay. The control unperfused ganglia
were removed oefore starting to perfuse the test ganglia. Under normal
conditions, both ganglia of the cat contain about the same amount of ACh
(Brown and Feldberg, 1936a). The increase in ganglion ACh content due to
the formation of surplus ACh was calculated by comparing the ACh content of
the perfused and the control ganglia; the results of these experiments are
shown in Fig. 2.
Ganglia that were perfused with eserine-Ch-Locke rapidly formed surplus
ACh, so that their total content almost doubled within 1 hr. Figure 2
compares the results of the present experiments using eserine-Ch-Locke with
the results of Birks and MacIntosh (1961) who perfused ganglia with eserine
plasma or left the natural circulation intact and injected large doses of '
TEPP; ganglia behaved similarly whichever medium was used. However, when
neostigmine was the anti-ChE, formation of surplus ACh was delayed; ganglion
ACh content increased by only 32% within the first hr, and perfusion for
120 min was required before the content doubled. 42
"~""""""'''''''''''''I'''II'I''I'''I'''''''''II'''"'_M'IIH· ... e -,;"""'''' _ -•.. _._ /,...... •. _/' ...... T__ -----: ... Cil l,l' A:i. ~./ ,". J. .,.'~ " ,,:of' , .. # ..../ l'" C "...... ' -• ....#...... ,,' C ...l' ," -0 '*...... ,' U ~# , oC ...... ,' U ...... ' ~ Sol' .' c ...... ,.' ~ ...... ,1 al / / X aC C) ,.' c .,'" ..• ..•a u oS 'fi. 20
o ' 30 90 120 150 lime (min)
FIG. 2. Rate of increase of ganglion ACh content due to the formation of surplus ACh by resting superior cervical ganglia perfused with Ch and an anti-ChE.
···@····0··· results from Birks and MacIntosh (1961); ganglia perfused with eserine-plasma or TEPP-natural circulation.
_.- •• _ .• _._. ganglia perfused with eserine-Ch-Locke; each point is the result of one experiment, except 60 min which is the mean of 2 experiments.
--6,-fj,-- gc:lnglia perfused with neostigmine-Ch-Locke; each point represents the mean + S.D. of at least 3 experiments.
x, X two experiments in which ganglia were perfused for 90 min with ambenonium-Ch-Locke. 43
Table l compares the ACh content of ganglia perfused for 20-150 min with neostigmine-Ch-Locke with the control unperfused ganglia. Column
5 of this table shows the P values obtained from at-test which assessed
the significance of the difference between the control and the perfused
ganglia. The ACh content of ganglia perfused for 20 or 30 min with
neostigmine-Ch-Locke did not differ significantly from their controls;
ganglia perfused for 60 min had increased their ACh content by 32% but
this difference was not quite statistically significan~while ganglia
perfused for 90-150 min had significantly more ACh than their controls.
The P value from a test for significant difference between the percentage
increase of ganglion ACh content at each successive time interval is given
in column 7 of Table 1; this comparison shows that in the presence of
neostigmine surplus ACh forms rapidly between 30 and 120 min, but that no
further increase occurred after 2 hr.
Ganglia that were perfused with ambenonium-Ch-Locke for 90 min
accumulated about as much surplus ACh as did ganglia that were perfused with
neostigmine (Fig. 2; Table II). The increase in ACh content of ganglia
perfused with ambenonium for times other than 90 min was not determined,
but it would appear that the rate of surplus ACh formation is not much
slower with ambenonium than it is with neostigmine.
It is clear from these experiments (Fig. 2; Table 1) that surplus ACh
formation can only about double. the ACh content of ganglia whatever anti-
ChE agent is used; however,• the time required to reach this maximum ACh
content is longer when neostigmine, rather than eserine, is the anti-ChE. e e
TABLE I. Formation of surplus ACh by ganglia perfused with neostigmine-Ch-Locke
(1) (2) (3) (4) (5) (6) (7) Time No. ACh Content (ng) ACh Content (ng) P Value % Increase in P Value (min) Expts. of Control Ganglion of Test Ganglion Calculated Ganglion ACh Calculated from t-Test (Mean ± S. D• ) (Mean ± S. D• ) from t-Test (Mean ± S.D.) one t:l:me vs. next time Test vs. Control
20 3 235.0 + 31.2 275.0 + 48.2 P-0.3 16.6 + 4.9 P)0.9 30 4 243.3 ± 70.9 280.7 + 71.0 P)0.5 16.1 + 5.9 P<0.05 60 3 255.0 + 51.9 334.8 + 63.8 P=O.l 31.8 + 8.5 P=O.OOI 90 4 300.0 + 53.4 495.0 + 71.4 .P ~ ~ 45 TABLE II. Formation of surplus ACh by ganglia perfused for 90 min with various anti-ChE agents. Anti-ChE agent % increase in ganglion ACh content 2 Eserine 961 ; 105 Neostigmine 65.8 + 7.8 3 Ambenonium 57, 514 1 eserine-Ch-Locke, 1 experiment. 2mean of experiments by Birks and MacIntosh (1961) using eserine-plasma, or TEPP-natural circulation. 3 mean + S.D. of 4 experiments. 4 individual values for 2 experiments. 46 B. Release of Acetylcholine by Nerve Stimulation into Different Anticholinesterase Agents The inhibition of AChE is necessary for the accumulation of surplus ACh. Because neostigmine and ambenonium were less effective .inhibitors of this AChE than was eserine, the ability of these drugs to inhibit functional AChE was tested. The results (Fig. 3) showed that eserine, neostigmine and ambenonium were equally effective in preserving ACh released by nerve stimulation. In 6 experiments, the amount of ACh released by a 4 min test stimulation (20/sec, 0.8 msec, 5 V) was measured during perfusion with each of the three anti-ChE agents in Ch-Locke. The results of these experiments are summarized in Fig. 3; there was no significant difference between the release of ACh into eserine and release into neostigmine (P>0.3) or ambenonium (P>0.8). In 2 of tnese experiments, the perfused ganglion and the control ganglion were assayed for ACh; test ganglia had not formed a significant amount of surplus ACh. Since functional AChE was as effectively inhibited by neostigmine or ambenonium as it was by eserine, the formation of surplus ACh must depend upon the inactivation of another AChE pool. C. Surplus Acetylcholine Formation in the Chronically Decentralized· Ganglion Although the available information suggested that surplus ACh was synthesized and stored in preganglionic nerve endings, more direct evidence was required. Therefore, the formation of surplus ACh in acutely (30 min) and chronically (1 week) decentralized ganglia was measured; the results of 46 B. Release of Acetylcholine by Nerve Stimulation into Different Anticholinesterase Agents The inhibition of AChE is necessary for the accumulation of surplus ACh. Because neostigmine and ambenonium were less effective inhibitors of this AChE than was eserine, the ability of these drugs to inhibit functional AChE was tested. The results (Fig. 3) showed that eserine, neostigmine and ambenonium were equally effective in preserving ACh released by nerve stimulation. In 6 experiments, the amount of ACh released by a 4 min test stimulation (20/sec, 0.8 msec, 5 V) was measured during perfusion with each of the three anti-ChE agents in Ch-Locke. The results of these experiments are summarized in Fig. 3; there was no significant difference between the release of ACh into eserine and release into neostigmine (P>0.3) or ambenonium (P>0.8). In 2 of these experiments, the perfused ganglion and the control ganglion were assayed for ACh; test ganglia had not formed a significant amount of surplus ACh. Since functional AChE was as effectively inhibited by neostigmine or ambenonium as it was by eserine, the formation of surplus ACh must depend upon the inactivation of another AChE pool. C. Surplus Acetylcholine Formation in the Chronically Decentralized Ganglion Although the available information suggested that surplusACh was synthesized and stored in preganglionic nerve endings, more direct evidence was required. Therefore, the formation of surplus ACh in acutely (30 min) and chronically (1 week) decentralized ganglia was measured; the results of 47 r" -~.. _. ---"------ " 50 .-c -a 30 •en •a GIi:• 20 ..c U CC 10 FIG. 3. The release of ACh from perfused sup~rior cervical ganglia during preganglionic nerve stimulation (20/sec, 0.8 msec, 5 V, for 4 min); ganglia perfused with Ch-Locke containing either neostigmine (stippled column) , eserine (hatched column) or ambenonium (black column). Each column represents the mean ± S.D. of 6 experiments. 47 50 - Q) 30 "en a Q) Q) 20 D: ..c U c( 10 ...... o ...... •••••••• ...... o ...... ••••••••• ...... o ...... •••• • ••• . . FIG. 3. The release of ACh from perfused superior cervical ganglia during preganglionic nerve stimulation (20/sec, 0.8 msec, 5 V, for 4 min); ganglia perfused with Ch-Locke containing either neostigmine (stippled column), es erine (hatched column) or ambenonium (black column). Each column represents the mean + S.D. of 6 experiments. 48 these experiments showed that chronicaily decentralized gang lia did not synthesize surplus ACh. These results are shown in Table III. In 2 experiments acutely decentralized ganglia were perfused with eserine-Ch-Locke;. their ACh content increased by 75% and 89% as expected. Chronically decentralized (unperfused) gang lia contained less than 40 ng ACh and had therefore lost about 85% of their ACh; this is consistent with the results of other workers (e.g. MacIntosh, 1938; Feldberg, 1943; Banister and Scrase, 1950; Friesen et al., 1967). When chronically decentralized ganglia were perfused for 60 min with eserine-Ch-Locke, their ACh content was not significantly (P>D.6) altered. If any surplus ACh formed in the decentralized ganglia, it was only about 3 ng, which was insignificant compared to the 240 or 210 ng that accumulated in ganglia whose preganglionic innervation was intact. These experiments show that the synthesis of surplus ACh occurs in the preganglionic nerve endings and not in the adrenergic cell body. D. Labelling of Surplus Acetylcholine The results of the experiments reported in section B above, as weIl as those of Birks and MacIntosh (1961), suggested that an active ganglion released as much transmitter when surplus ACh is allowed to form as it does when no extra ACh is forming; thus surplus ACh appears not to be available for immediate release by nerve impulses. Whether surplus ACh can mix with depot ACh was not known,and this could only be determined if a method could be devised whereby surplus ACh could be labelled. Birks and MacIntosh (1961) showed that a supply of Ch in the perfusion fluid was necessary for surplus ACh formation by eserinized ganglia. It 49 TABLE III. Accumulation of surplus ACh by normal and chronically (1 week) decentralized ganglia during perfusion with eserine-Ch Locke for 60 min. ACh content (ng) Experiment Perfused Non-perfused Surplus ACh formed (ng) A. Both gang lia chronically decentralized 1 "'30 "'30 "'0 2 "'18 "'15 "'3 3 "'25 "'40 "'0 B. Both gang lia acutely decentralized 4 510 270 240 5 490 280 210 50 therefore seemed likely that if the Ch source was radioactively labelled, labelled surplus ACh would accumulate. This was tested in 4 experiments by perfusing resting superior cervical ganglia with 3H-Ch-eserine Locke for 60 min; in aIl the experiments, 3H- ACh could be identified in the extracts of perfused ganglia. The total ACh content of the control and of the perfused ganglia was measured by bio-assay so that the total amount of surplus ACh formed could be determined. The 3 H-ACh in the test ganglia extract was separated from other labelled material (see Methods and Materials) and dividing the radioactivity (DPM) on ACh by the total surplus ACh formed (ng) gave the specifie activity of surplus ACh; by comparing this specifie activity with the specifie activity of the 3H- Ch used (correcting for the change caused by the acetylation), the proportion of the formed surplus ACh that had been labelled could be calculated. The assumption was made that aIl of the labelled ACh was surplus ACh, and this is not quite valid for some turnover of depot ACh will occur even in the absence of stimulation (Collier and MacIntosh, 1969); however, this assumption did not alter the results appreciably (see below). Table IV contains the resultsof these experiments. The mean ACh content of control ganglia was 349 ng, and that of the perfused gang lia was 640 ng; thus ganglion ACh content had increased by 83% because of the formation of surplus ACh. Of the 291 ng of surplus ACh formed, 139 ng was labelled; thus 49% of the surplus ACh formed had been labelled. The specifie activity of the surplus ACh accumulated by these gang lia was about 375 DPM/ng; if surplus ACh had mixed freely with depot ACh, the specifie activity of ganglion ACh (based on total ACh content - i.e. depot and surplus ACh) would be about 170 DPM/ng. On the basis of these values, e e TABLE IV. The labelling of surplus ACh in superior cervical ganglia; ganglia were perfused at rest for 60 min with eserine-3H-Ch-Locke. Total ACh content Experiment Total Labelled % Surplus ACh No. Control ganglion Test ganglion surplus ACh surplus ACh labelled (ng) (ng) (ng) (ng) 1 330 600 270 90 33.3 2 285 510 225 160 71.1 3 360 650 290 128 44.1 4 420 800 380 176 46.3 Mean + S.D. 348.8 + 56.6 640.0 + 121.4 291.2 + 65.1 138.5 + 37.9 48.7 + 15.9 .....\J1 52 it was predicted that: (a) if nerve stimulation released ACh from a single pool (i.e. if surplus and depot ACh mix free1y), then the re1ease of 5 ng of this ACh (about 850 DPM) would be detected easi1y; (b) if surplus ACh does not mix with depot ACh, and if nerve stimulation can re1ease surplus ACh, then the release of 2 ng (about 750 DPM) of this ACh cou1d be detécted easi1y; (~) if surplus ACh does not mix with depot ACh, and if nerve stimulation do es not release surplus ACh, then on1y un1abe11ed ACh would be detected. The next section reports the resu1ts of such experiments. E. Fai1ure of Nerve Stimulation to Release Surplus Acetylcholine: Perfusion with Eserine-Cho1ine-Locke Surplus ACh of the cat's superior cervical ganglion was 1abel1ed as described above (section D) and at the end of the loading perfusion, the perfusion fluid was switched to one containing eserine-un1abe11ed Ch-Locke; perfusion was continued for a second 60 min period. The effluent was col1ected in 2 min samp1es and an a1iquot of each was counted for radio activity. The pregang1ionic sympathetic nerve was stimu1ated between the 14th and the 30th min of this washout perfusion, and the samp1es col1ected during stimulation were assayed (by bio-assay) for their total ACh content as weIl as for radioactivity. These experiments showed that 1abe11ed surplus ACh is not re1eased by nerve stimulation. The resu1ts of a typica1 experiment are shown in Fig. 4. At first radioactivity in the ganglion effluent was high but it soon began to 1eve1 off. Stimulation of the pregang1ionic sympathetic nerve (20/sec, 0.8 msec, 5 V) for 16 min fai1ed to re1ease 1abe11ed materia1, a1though a total of 130 ng of un1abe11ed ACh was re1eased during this test period. Simi1ar 53 '0\ 70 .E ..,E 50 1". '. i c:' li.. 0 ;ë -III To~ol ACh Rel.a ••d ~ ..: Do .1: Cl 40 U c 130 nll \ o 10 20 30 40 50 60 \ ~ Tlm. Cmln} .~ STlM.20Hz 10 ,~ • STIM. 20~~-_."i''''''.'''' ...... , ...... _ O~------~IO~~~~2~O~~~~30~------"~O------~5~O------6~O- Tim. {min} FIG. 4. Lower: The effect of nerve stimulation upon the efflux of radio activity from a catIs superior cervical ganglion perfused with eserine-Ch Locke; the ganglion's surplus ACh pool had been labelled by perfusing the resting ganglion for 60 min with eserine-3H-Ch-Locke. Preganglionic nerve stimulation (20/sec, 0.8 msec, 5 V) was throughout the hatched bar. Upper insert: Same experiment; the effect of nerve stimulation on the release of ACh (measured by bio-assay). 54 resu1ts were obtained from 4 other eXperiments in which the frequency of nerve stimulation (3, 5 or 10/sec) was c10ser to the physio10gica1 range; a11 these resu1ts are shown in Tab1eV. Nerve stimulation a1ways re1eased ACh but never re1eased a significant amount of radioactivity; in some of the experiments there was a sma11 release of 1abe11ed material in the first samp1e col1ected during nerve stimulation, but it cou1d not be determined with certainty whether or not this was rea1. At the end of a1l experiments the ganglia were removed and extracted for bio-assay and for separation of ·labe1led ACh from other radioactive material. The resu1ts of these analyses are summarized in Table VI. The perfused gang1ia had increased their ACh content by 96% and this confirms that the amount of surplus ACh remains approximate1y constant during a second hr of perfusion with eserine and Ch (see a1so Fig. 2). Of the 249 ng of surplus ACh in these ganglia, on1y 81 ng or 34% was 1abe11ed; thus, the percentage of surplus ACh that was 1abe1led had fal1en from 49% at the end of the 10ad (section D above) to 34% at the end of this washout perfusion. This turnover of surplus ACh is i1lustrated in Fig. 5 which shows the total amount of surplus ACh after the 1 hr loading perfusion was not significantly different from that at the end of the 1 hr washout perfusion, but the specific activity of this surplus ACh fe1l (P With the reduced amount of label1ed surplus ACh present at the end of the experiment, the specifi.c activity of surplus ACh was 257 DPM/ng and therefore its specific activity at the time of stimulation must have been somewhere between this value and that of 375 DPM/ng (section D above). Nerve stimulation released 40-75 ng in the first 4 min collection period, and it is c1ear that surplus ACh could not have contributed more than a few e e TABLE V. The effect of nerve stimulation upon the efflux of radioactivity and upon the release of ACh from superior cervical ganglia perfused with eserine-Ch-Locke; the ganglia's surplus ACh pool had been labelled. Frequency of ACh released (ng) (bio-as say) DPM Released Experiment stimulation first 2 min (pulses/ sec) First 4 min Total (16 min) 1 3 45 95 0 2 5 40 80 ~100 3 10 60 110 ~200 4 20 70 130 0 5 20 75 120 ~300 VI VI e e TABLE VI. Labelled and unlabelled ACh in superior cervical ganglia that had been perfused for 60 min with eserine-3H-Ch-Locke and then for 60 min with eserine-unlabelled Ch-Locke. Total ACh content Experiment Total surplus Labelled surplus % surplus ACh No. Control ganglion Test ganglion ACh (ng) ACh (ng) Labelled content (ng) content (ng) 1 260 575 315 90 28.6 2 280 550 270 80 29.6 3 300 570 270 84 31.1 4 220 430 210 73 34.8 5 240 420 180 78 43.3 Mean + S.D. 260.0 + 31.6 509.0 + 77.3 249.0 + 53.6 81.0 + 6.4 33.5 + 5.9 \JI 0\ 57 ------~-- ~---_. - ----_.~---_._---_._._--_._--- ' ' 1 100 1, i i 80 400 -- 1 CIl ...c: 1 1 c :lE t -! a. c Q 0 1 40 300 1 oC" r ~ 1 C .c u 1 ~ 1 CIl cc c .. 1 CI :J CIl 30 200 Q, ! .: .. 1 :J CIl ... .. 1 CI ëi ! 1 .5" .( ! !li!. 20 100 ui 0-10--- FIG. 5. Turnover of surplus ACh. Left: The percentage increase in ganglion ACh content by ganglia perfused at rest for 60 min with eserine-3H-Ch-Locke (hatched column, 4 experiments) and ganglia perfused for an additional hour with eserine unlabelled Ch-Locke (stippled column, 5 experiments). Each column represents the meari + S.D. Right: Same experiments; the specifie activity of surplus ACh (DPM/ng) in ganglia perfused at rest for 60 min with eserine-3H-Ch-Locke (hatched column) and in ganglia perfused for an additional hour with eserine unlabelled Ch-Locke (stippled column). 58 ng to this release without being detected. The absence of an increased efflux of labelled material during stimulation of these ganglia shows that the loading procedure did not label a significant amount of depot ACh; these experiments also show that surp1us ACh does not mix with the depot ACh pool and confirm that it cannot be mobilized for release by nerve stimulation. In addition, these experiments show that surplus ACh continues to turnover even when its total amount is constant. These ganglia accumu1ated about 249 ng of surplus ACh and it would be expected that after an br's treatment with 3H- Ch and eserine about half of this (section D above) wou1d be labelled, i.e. 125 ng; when the ganglia were then perfused for a second hr with unlabelled Ch and eserine, they retained their 249 ng of surp1us ACh, but only 81 ng remained labelled. This loss of 44 ng of labe11ed ACh was presumably accompanied by a loss of an equal amount of unlabe1led surp1us ACh, and thus the total turnover would be 88 ng or 35%. This turnover of surplus ACh during its steady state (about 1.5 ng/min) is about 1/3 of its rate of synthesis in a fresh ganglion. F. Failure of Nerve Stimulation to ReleaseSurplus Acetylcholine: Perfusion with·Eserine-Hémicholinium-3~Locke In section E above it was shown that surplus ACh was not re1eased by nerve stimulation when ganglia were supplied with Ch. The experiments described in this section determined if gang lia could draw upon surp1us ACh when its depot pool was being depleted. The results of these experiments indicate that surplus ACh cannot be used as a source of transmit ter substance even in emergency conditions. 59 In these experiments, the procedure was the same as described in section E above, except that in the second hr of perfusion, the medium was switched to eserine-HC-3-(Ch-free)-Locke, and the duration of nerve stimulation was extended for an additional 14 min (i.e. for a total of 30 min). The results of a typical experiment are shown in Fig. 6. As in the previous experiments, nerve stimulation (20/sec, 0.8 msec, 5 V) failed to release labelled surplus ACh but effectively released unlabelled ACh. Four other experiments at the same frequency of stimulation gave similar results, and aIl these results are shown in Table VII; nerve stimulation released at most only a few DPM. At the end of aIl these experiments, the ganglia were removed, and their ACh was extracted for bio-assay and for the separation of labelled ACh from other labelled material. The results of these analyses are in Table VIII. In calculating the amount of surplus ACh in perfused ganglia at the end of the experiment, the ACh content of ~he control ganglion was subtracted from the sum of the amount of ACh released during nerve stimulation and the amount remaining in the test ganglion at the end of the experiment. This was necessary since in the presence of HC-3 no synthesis of ACh can occur; in the experiments in section E above, test stimulation was in the presence of eserine-Ch-Locke, where released ACh is replaced by synthesis and no such calculation was required. The sum of ACh released from, and ACh remaining in the perfused ganglia totalled 458 ng, while the ACh content of control ganglia was 302 ng; the test ganglia thus contained 52% more ACh due to the presence of 156 ng of surplus ACh. If it is assumed that at the end of the loading perfusion the test ganglia had incrëased their total ACh content by at least 85% (by forming 257 ng surplus ACh) , it is clear that in the second hr of perfusion (with 60 ~ . 60 'ë" 50 00 • "0 -•;; • Total ACh Relea.ed ~ ';' 40 m 170 ng la ..s ~ .. u ~ ...a '" 30 10 20 30 40 50 60 20 o ~ Tlme (min) 5T1M. 20 Hz \ \. 10 ,., ...... -.-.-.-- 10 _ 20 30 40 .--50 60 o Time (min) --STiM. 20 Hz ___- _____ ---.1 FIG. 6. Lower: The effect of nerve stimulation upon efflux of radioactivity from a cat's superior cervical ganglion perfused with eserine-HC-3-Locke; the ganglion's surplus ACh pool had been labelled by perfusing the resting ganglion for 60 min with eserine-3H-Ch-Locke. Preganglionic nerve stimulation (20/sec, 0.8 msec, 5 V) was throughout the hatched bar. Upper insert: Same experiment; the effect of nerve stimulation on the release of ACh (measured by bio-assay). 61 TABLE VII. The effect of nerve stimulation upon the efflux of radioactivity and upon the release of ACh from superior cervical ganglia perfused with eserine-HC-3-Locke; the ganglia's surplus ACh pool had been 1abel1ed. ACh released (ng) (bio-assay) Experiment DPM released First 6 min Total (30 min) first 2 min 1 57 170 0 2 75 150 "'800 3 48 100 "'600 4 56 105 "'300 5 65 140 0 Mean + S .D. 60.2 + 10.2 133.0 + 29.9 340.0 + 357.7 61 TABLE VII. The effect of nerve stimulation upon the efflux of radioactivity and upon therelease of ACh from superior cervi·cal ganglia perfused with eserine-HC-3-Locke; the ganglia's surplus ACh pool had been labelled. ACh released (ng) (bio-assay) Experiment DPM released First 6 min Total (30 min) first 2 min 1 57 170 0 2 75 150 ::0800 3 48 100 =600 4 56 105 =300 ·5 65 140· 0 Mean + S.D. 60.2 + 10.2 133.0 + 29.9 340.0 + 357.7 e e TABLE VIII. Labelled and unlabelled ACh in superior cervical ganglia that had been perfused for 60 min with eSE!rine-3H-Ch-Locke and then for 60 min with eserine-HC-3-Locke. Total ACh content Experiment Control ganglion Test ganglion Total surplus ACh Labelled surplus ACh % surplus ACh No. content content + release (ng) (ng) labe11ed (ng) (ng) 1 260 390 130 60 46.1 2 315 510 195 69 35.4 3 360 550 190 75 39.5 4 295 405 110 65 59.0 5 280 435 155 43 27.7 Mean + S.D. 302.0 + 38.1 458.0 + 69.1 156.0 + 36.9 62.4 + 12.1 41.5 + 11.8 0\ N 63 HC-3-eserine-Locke) the tissues lost 39% of this surplus ACh even though eserine was sti~l present. This would be expected from the results described in section E above which showed that surplus ACh turns over even when its total amount is constant; HC-3 by inhibiting synthesis would prevent continued formation of surplus ACh and result in its loss. The rate of loss of surplus ACh in these experiments was about.l.7 ng/min (a total of 100 ng lost in 1 hr) and this is similar to the estimated rate oi turnover (1.5 ng/ min) reported in section E above. In these experiments that us~d HC-3, the proportion of labelled ACh in the surplus ACh remaining at the end of the experiment (42%) was not significantly different (P>0.4) from the proportion of labelled surplus ACh at the end of the load (49%, section D above). This would be expected because any loss of surplus ACh would affect equally the labelled and unlabelled ester, and thus keep their proportion constant. Because the loss of ACh in these experiments did not alter the specifie activ1ty of surplus ACh (about 320 DPM/ ng at the end of the experiment) they are perhaps a more sensitive test for the release of surplus ACh during stimulation than are the experiments in section E above; less than 2 or 3 ng of labelled.ACh could have contributed to release in the present experiments without being detected. The failure of labelled surplus ACh to appear in the effluent supports the findings reported in section E above that sur.plus ACh does not mix with releasable transmitter; in addition, it .appears that the depot pool cannot draw upon surplus ACh as a source of transmitter even when stimulation in the presence of HC-3 depletes the depot pool. Finally, these experiments confirm that there is a turnover of surplus ACh. 64 + G. ~elease of Surplus Acetylcholine by High K -Locke + - High K has long been known to releasedepot ACh from the cat's superior cervical ganglion (Brown and Feldberg, 1936a) and the present experiments show that high K+ also releases surplus ACh. In these experiments surplus ACh was labelled as described in section D above except that in some experiments DFP was the anti-ChE; perfusion fluid was then switched to Ch-Locke containing either DFP or eserine. Effluent collected during the first 20 min of this perfusion was discarded; samples were then collected every 2 min and perfusion was changed to a solution containing 56 mM KCI for 6 min. The results of one experiment are illustrated in Fig. 7. When perfusion was changed to a high K+ medium, radioactive material was released. Four other experiments gave similar results although the amount of radioactivity released by high Kwas+ smaller; these results are in Table IX. The values headed "calculated" were determined from the amount of extra radioactivity released during perfusion with high K+, and those values headed "measured" were measured as labelled ACh by the reineckate precipitation procedure. In one experiment, labelled ACh in an aliquot of + the ganglion effluent collected during perfusion with high Kwas determined by both methods; its content of labelled ACh measured directly agreed within 7% with the value calculated from the extra radioactivity released. Thus the only radioactive material released by high Kwas+ ACh. The total amount of surplus ACh released (i.e. labelled and unlabelled) cannot be measu~ed accurately in the present experiments; since approximately 49% of surplus ACh was labelled after the loading perfusion (section D above) and about 34% was labelled after the second hr of perfusion with eserine-Ch- Locke (section E above),it is probable that the total amount of surplus ACh 65 . - ._--- 1 i 1 1 1 • ·1 1 30 C") 20 1 • 0 -~ ~ \. D. .-. C f'· '. 10 B \ 1 .-.,.,.... 0\ :\ o 30 40 l,im e (min) ~':. FIG. 7. The effect of high K+ (56 mM) perfusion upon the efflux of radioactivity froma catIs superior cervical ganglion perfused with DFP Ch-Locke; theganglion's surplus ACh pool had been labelled by perfusing the resting ganglion for 60 min with DFP-3H-Ch-Locke. "B" represents a blank injection of 0.2 ml Locke through thetubing leading from the high 1(+ perfusion bottle; perfusion with high ICI- throughout the hatched bar. 66 TABLE IX. The effect of high K+ perfusion (6 min) upon the eff1ux of radioactivity from superior cervical gang1ia whose surplus ACh pool had been 1abe11ed. Labe11ed surplus Labe11ed surplus ACh re1eased DPM re1eased· ACh·re1eased (ca1cu1ated) (measured) (ng) (ng) 10,000 13 4,800 6 6,700 9 5,900 8 31,500 41 44 67 released was 2-3 times the amount of labelled surplus ACh released (i.e. the values in Table IX). + . The radioactivity released by high K in these experiments could not have come from the depot ACh pool, for the experiments above (section E) demonstrated that the loading perfusion did not label the releasable depot ACh. H. Release of Surplus Acetylcholine by Acetylcholine or by Carbachol The experiments described in this section tested the effects of ACh or carbachol upon the release of surplus ACh from superior cervical ganglia; the results show that both drugs effectively release surplus ACh. Surplus ACh was labelled as described in section D above, except that in most experiments DFP was the anti~ChE; perfusion was then switched to Ch-Locke containing either DFP or eserine. .The ganglion effluent during the first 20 min of this second hr of perfusion was discarded, and it was then collected every 2 min; perfused ACh,and perfused or injected carbachol were then tested for their ability to release radioactivity. a) Release of surplus acetylcholine byacetylcholine. The results of a typical experiment which tested the release of surplus AChby perfused ACh are illustrated in Fig. 8. Stimulation of the preganglionic sympa the tic nerve (5/sec 5 0.8 msec, 5 V) failed to release labelled material, but perfusion with ACh (0.5 or 15 ~g/m1) for 6 min released radioactivity and this release was dose dependent. Similar results were obtained in 10 other tests, 3 using eserine and 7 using DFP as the anti-ChE, although the amount of labelled ACh released was variable. AlI of these results are shown in Table X. In most experiments, the amount of labelled surplus ACh released 68 -. - ._--- -.-- ._- ----~------i i 20 . • ! 15 M b.... ->< 10 ~ D O 5 " j/\ /\ '--...... -_.-.-. -, ~ f ~f .--, 5Hz B B -~5Hz ----- o 20 lime (min) 1 -' FIG. 8. The effect of nerve stimulation and of perfused ACh (0.5 or 15 ~g/m1) upon the eff1ux of radioactivity from a cat's superior cervical ganglion perfused with DFP-Ch-Locke; the gang1ion's surplus ACh pool had been labe11ed by perfusing the resting ganglion for 60 min with DFP_3H- Ch-Locke. "B" represents a b1ank injection of 0.2 ml Locke through the tubing 1eading from the ACh perfusion bott1es; ACh perfusion or pregang1ionic nerve stimulation (5/sec, 0.8 msec, 5 V) throughout the batched bars as indicated. e e TABLE X. The effect of ACh perfusion (6 min) upon the efflux of radioactivity from superior cervical gangiia whose surplus ACh pool had been labelled. Labelled surplus . Labelled surplus ACh concentration ACh released ACh released And-ChE agent DPM released Ülg/ml) (calculated) (measured) (ng) (ng) A. Eserine 0.5 5,700 7 1.0 5,700 7 1.5 14,000 18 16 B. DFP 0.5 5,900 8 0.5 10,000 13 1.0 15,000 20 20 1.0 8,500 Il 13 1.0 21,000 27 1.0 5,900 8 1.0 6,600 9 2.0 6,600 9 15.0 26,000 34 29 0\ \0 70 was calculated from the extra radioactivity collected during perfusion with ACh. In 4 experiments, the labelled .material in an aliquot of the ganglion effluent was separated by the reineckate precipitation test. These values are entered in Table X under the columns headed i'calculated" and "measured", respectively. The two estimates of the amount of labelled surplus ACh released by ACh agreed within 17%, showing that the whole of the extra radioactivity released was labelled ACh. Table X shows that ACh released surplus ACh when tested in the presence of either eserine or DFP. One experiment tested the release of labelled surplus ACh by ACh (O.S pg/ml, 6 min) first in the presence of DFP and then in the presence of eserine; labelled surplus ACh had been formed in DFP. The results of the experiment are shown in Fig. 9; an equal amount of labelled surplus ACh was released by ACh in both media. The release of surplus ACh by ACh in some of these experiments was transient where most of the release was within the first 2 min of ACh perfusion (e.g. Fig. 9); in others, the effect of ACh lastedlonger (e.g. Fig. 8). The total amount of surplusACh released by ACh could not be measured accurately in the present experiments but could be estimated to be 2-3 times the amount of labelled surplus ACh released (see section G above). b) Releaséof"surplus"àéetylcholineby carbachol. Figure 10 shows the results of a typical experiment which tested the release of surplus ACh by injected or perfused carbachol. Injected carbachol (0.5 or 1.0 pg) released labelled surplus ACh and this effect increased with increased dose; however, perfused carbachol (1.0 or 15 pg/ml, 4 min) failed to release a significant amount of radioactivity. 71 , j ! r • 1 ! 1 ! 1 \ 1 1 ! 1 ! 1 1 \t'· \ • 1 B '.-.f 1 B i 1 ! lime (min) " ._. __ ___ .______. __ "__ .___ .___ .J FIG. 9. The effect of perfused ACh (0.5 pg/ml) upon the efflux of radioactivity from a cat's superior cervical ganglion whose surplus ACh pool had been labelled by perfusing the resting ganglion with DFP_3H-Ch Locke; perfusion with ACh in DFP-Ch-Locke throughout period indicated by cross-hatched bar and perfusion with ACh in eserine-Ch-LQcke throughout period indicated by hatched bar. "B" represents a blank injection of 0.2 ml Locke through the tubing leading from the ACh perfusion bottles. 72 '. 15 • 10 C"') '0... >< ~ A. Q 5 • B C.P. C.P. ~_.-. \ 1.0~.~lml 15.0~~/ml .-. ct ., ~ O.5JJ9 .-.-.'. -.---.-.-.-- ' ...... , , 30 40 50 60 lime (min) FIG. 10. The effect of injected (0.5 or 1.0 ~g) or perfused (1.0 or 15 ~g/m1) carbacho1 upon the eff1ux of radioactivity from a catis superior cervical ganglion perfused with DFP-Ch-Locke; the gang1ion's surplus 'ACh pool had been 1abe1led by perfusing the resting ganglion for 60 min with DFP_3H-Ch-Locke. "B" represents a b1ank injection of 0.2 ml Locke through the tubing through which carbacho1 was 1ater injected. "c" represents an injection of carbachol in 0.2 ml Locke. "C.P." represents carbacho1 perfusion throughout the hatched bar. 73 Other experiments tested injected carbachol (0.5-2.5 Vg) or perfused carbachol (1.0-30 Vg/ml, 6 min), and the results are in Table XI. Injected carbachol effectively released surplus ACh in aIl experiments but when perfused was effective only at high concentrations. In one experiment, the amount of labelled surplus ACh released by injected carbachol was measured by selective precipitation tests and the value compared to that calculated from the extra radioactivity released; the two values agreed within 18%, showing that the only radioactive material released by carbachol was ACh. The accurate determination of total surplus ACh released by carbachol cannot be obtained by the present procedures; however as in section Gabove, the total surplus ACh released must have been 2-3 times the amount of labelled surplus ACh released. The results of this section demonstrated that exogenous ACh or carbachol released surplus ACh. 1. Ganglion Stimulation by Carbachol in the Preséncéor Absence of Surplus Acetylcholine The above experiments (section H) demonstrated that injected carbachol released surplus ACh from superior cervical ganglia and the present experiments tested the contribution of this evoked release to drug-induced ganglion stimulation. Ganglia were perfused with Ch-Locke and the submaximal ganglion stimulant effect of injected carbachol (1 Vg) was measured every 10 min by recording nictitating membrane contraction. Perfusion was then changed to DFP-Ch-Locke, and the tests were continued; in this medium the ganglion would accumulate surplus ACh so that its content wou Id about double within an hr. e e TABLE XI. The effect of injected or perfused (4 or 6 min) carbacho1 upon the efflux of radioactivity from superior cervical ganglia whose surplus ACh pool had been 1abe1led. Labe11ed surplus Labe11ed surplus ACh released ACh released Carbachol dose DPM released (ca1culated) (measured) (ng) (ng) A. Injected llg 0.5 10,000 13 1.0 12,500 16 2.5 20,000 26 22 B. Perfused llg/ml 1.0 0 0 10 0 0 15 0 0 15 1,300 <2 30 3,500 <5 "~. 75 A typical record from one of these experiments is shown in Fig. 11; ganglion stimulation by carbacholwas the samewhen surplus ACh was forming as it was when there was no surplus ACh. This shows that the surplus ACh released by carbachol does not augment the ganglion stimulant effect of . this drug. e e _. ___ .. --"4 ______..._ .. ,.. - ______--.---- ... --.-____. ___ ._._~_ .• --_._~, --,~---,------ A 4- 2 "'"D ~kkk -c t t t 1 t .,.0 3 J'g 1J'g 1J'g 1J'g l..,g -c r- B • 4 l- I 2 v·1. 0 t\ LkkLI t t . t t 1J'g 1J'g 1J'g 1J'g 1 1 1 min ~ t .,._._~----,------~~ FIG. Il. Contractions of the cat'snictitating membrane induced by carbachol injections; (A) ...... during perfusion with DFP-(Ch-free)-Locke and (B) during perfusion with DFP~Ch-Locke. Carbachol 0\ (in 0.2 ml Locke) was injected every 10 min. 77 IV. DISCUSSION 78 A. Formation of Surplus Acetylcholine Surplus ACh is the extra ACh that ~he cat superior cervical ganglion synthesizes and stores in the presence of Ch and an anti-ChE. The formation of this pool of ACh suggests that the synthesis and break down of ACh is continual1y occurringi the addition of an anti-ChE is needed to unmask this process and a1low its accumulation. It is not yet entirely c1ear where surplus ACh is stored. Chronica11y decentra1ized ganglia lose 90% of their ChAc within 1 week (Hebb and Waites, 1956) and this shows that almost al1 of the ganglion's ChAc is in the presynaptic nerve endings; as this enzyme is required for ACh synthesis, it is probable that surplus ACh is synthesized in the pregang1ionic nerve endings. This suggestion was confirmed in the present study by experiments which showed that gnng1ia whose preganglionic nerve had been al1owf:,·d to degenerate were unab1e t.o synthesize any surplus ACh. The requirement for both an anti-ChE agent and a source of Ch for the accumulation of surplus ACh by ganglia has been known since the work of Birks and MacIntosh (1961), and the results of the present experiments, when eserine-Ch-Locke was the perfusion medium, are not too different from those of Birks and MacIntosh who perfused gang lia with eserine-plasma. In addition, the present experiments show that the accumulation of surplus ACh is de1ayed considerab1y when the anti-ChE used is neostigmine or ambenonium instead of eserine. The amount of ACh collected from an active ganglion was the same when perfusion was with eserine, neostigmine or ambenonium, and this shows that these agents are equally effective in inhibiting functional AChE (i. e. the 79 AChE responsible for destroying neuronally released ACh). This confirms the results of Emmelin and MacIntosh (1956) who had shown previously that release into neostigmine or eserine-containing solution was about the same; they did not test release into both agents in the same preparation, and in most of their experiments ACh turnover was sub optimal due to lack of Ch in the perfusion medium. Because eserine, neostigmine and ambenonium inhibited functional AChE equally weIl, yet surplus ACh formed less easily in neostigmine or in ambenonium than in eserine, the formation of surplus ACh must depend upon the inactivation of an AChE pool other than functional AChE. Koelle and Koelle (1959) demonstrated the existence of two AChE pools: one (functional AChE) is easily accessible to the extracellular space and this enzyme is probably responsible for ACh destruction; the second enzyme pool (residual AChE) is intracellular and probably a "reserve" store. It seems likely that this reserve AChE has to be inactivated to allow the accumulation of surplus ACh. The most likely explanation for the delayed formation of surplus ACh when neostigmine or ambenonium, rather than eserine was the anti-ChE agent used, is that eserine which has a high lipid solubility can penetrate the nerve ending more easily than can neostigmine or ambenonium which have low lipid solubility (Burgen and Chipman, 1952; Koelle, 1957). This suggestion is supported by previous workers who showed that eserine can more readily penetrate the axon than can neostigmine or ambenonium (Bu1lock et al., 1946; Feld et al., 1948; Koelle, 1957). 80 B. Turnover of Surplus Acetylcholine Although the formation of surplus ACh in the cat's superior cervical ganglion was delayed when perfusion was with neostigmine rather than with eserine, the amount of surplus ACh eventually formed in the presence of either agent was the same and ganglionic ACh content doubled in either medium. If perfusion was continued after the full complement of surplus ACh had formed, no further increase in ACh content was observed. This suggests that ganglia can only double their ACh content. A similar phenomenon has been described recently for the mammalian skeletal muscle (Potter, 1970). In these experiments, Pot ter incubated the rat phrenic nerve diaphragm in medium containing eserine and l4c_Ch ; with intermittent nerve stimulation during the 1 hr incubation the ACh content of the muscle nearly doubled. No significant increase in the surplus ACh pool was seen after the first hr. Similar experiments have also been described for brain tissue; in vitro, minced or sliced brain incubated in a suitable medium will increase its ACh content to a maximum level at which it remains constant (Stedman and Stedman, 1937; Mann et al., 1939; Polak, 1969; Sharkawi and Schulman, 1969); in vivo, brain ACh content can increase in certain situations, but again there seems to be a ceiling level beyond which it cannot increase further (Tobias, Lipton and Lepinat, 1946; Torda, 1953; Giarman and Pepeu, 1962; Cross land and Slater, 1968). This upper 1imit for ACh content might reflect: (a) an equilibrium condition when formation and breakdown occur at equal rates. This is unlikely in the superior cervical ganglion because the same upper limit of ACh content is reached with the irreversible anti-ChE agent TEPP as is reached with the reversible 81 agent eserine (Birks and MacIntosh, 1961). (b) Inhibition of synthesis as a resu1t of end-product accumulation. There is in vitro evidence that ACh can inhibit ACh synthesis (Kaita and Go1dberg, 1969), but the maximum inhibition was somewhat 1ess than 50% even with very high ACh concentrations. (c) A saturation of ACh binding sites so that synthesized ACh can no longer be stored. The present experiments suggest that both (b) and (c) contribute to the maintenance of a steady ACh 1eve1 in the superior cervical ganglion. The gang1ion's surplus ACh was 1abe11ed by perfusing the resting preparation with eserine and 3H-Ch-Locke. These gang1ia increased their ACh content by formingsurp1us ACh, about ha1f of which was 1abe11ed; t~e gang1ion's endogenous Ch (Friesen et al., 1967) is probab1y the precursor for the un1abe11ed surplus ACh synthesized. When the 10ading perfusion was fo110wed by an hr's washout perfusion with un1abe11ed Ch and eserine, the total amount of surplus ACh remained constant, but its specifie radioactivity fe11 by 31%. This turnover of surplus ACh was confirmed by experiments which showed that if gang1ia with a full complement of surplus ACh were perfused with HC-3-eserine~(Ch free)-Locke, they lost 39% of their surplus ACh. The turnover of surplus ACh was measured to be about 1.5 ng/min by either method. Under optimal conditions, surplus ACh forms at a rate of about 4 ng/min; thus in the second hr of perfusion with eserine and Ch, synthesis of ACh is inhibited by about 60%, but content is steady and binding must be limiting. 82 C. Release of Acetylcholine by Nerve Stimulation In ganglia whose surplus ACh had been labelled, preganglionic nerve stimulation during perfusion with eserine-unlabelled Ch-Locke failed to release labelled·material, although non-labelled ACh was released and labelled surplus ACh remained in the ganglia at the end of the experiment. The failure of nerve stimulation to release labelled surplus ACh in these experiments confirms the suggestion of Birks and MacIntosh (1961) that surplus ACh is not immediately available for release; it also shows that surplus ACh does not mix with the releasable transmitter pool. The specifie activity of surplus ACh at the end of these experi ments was 257 DPM/ng and therefore as little as 4-5% mixingof surplus ACh with depot transmitter would have been detected by the present methods. If depot ACh is in vesicles and surplus ACh is free in the cytoplasm, it is likely that the ACh concentration in the vesicles would be several times higher than the ACh concentration in the cytoplasm; under the conditions of the present experiments, depot and surplus ACh are about equal in amount but the volume of vesicles must be much smaller than the volume of the cytoplasm. It could therefore be argued that the absence of mixing of the two pools of ACh may be due to the failure of surplus ACh to move against a concentration gradient into the depot pool. However, the large amount of surplus ACh which is synthesized in these ganglia was not used during stimulation in the presence of HC-3 when 45% of the ganglion depot pool was depleted; in this situation the concentration gradient of ACh was presumably reversed at least for many of the vesicles, but surplus ACh did not enter the depot pool. Therefore, it appears that surplus ACh is incapable of mixing with releasable transmitter, nor can it become available 83 for re1ease when gang1ia are in "emergency" conditions and synaptic transmission is fai1ing. Potter (1970) demonstrated that during brief nerve stimulation the rate of re1ease of ACh from the rat phrenic nerve-diaphragm was the same in the absence or presence of surplus ACh. However, during pro1onged nerve stimulation in the presence of HC-3, ACh re1ease was better maintained in those preparations which had accumu1ated surplus ACh than in those which had not, and Potter suggested that surplus ACh can contribute to the re1easab1e transmitter pool. Since diaphragm preparations which had accumu1ated surplus ACh re1eased more ACh spontaneous1y (perhaps by 1eakage from the cytop1asm) than did fresh preparations, the maintained re1ease in the presence of surplus ACh can a1ternate1y be exp1ained by the contribution of the increased spontaneous re1ease. Recent evidence indicating that ChAc is a soluble component of nerve ending cytop1asm (Fonnum, 1966, 1967) has suggested that ACh might be synthesized within the cytop1asm. New1y synthesized ACh wou1d then presumab1y be packaged into vesic1es when needed for synaptic transmission, or wou1d be re1eased from the ChAC synthesizing enzyme for destruction by AChE. An anti-ChE agent wou1d preserve this cytop1asmic ACh that is norma11y destined for hydro1ysis by residua1 AChE, and a110w the accumulation of surplus ACh. If the depot pool obtains its transmitter from a site of synthesis in the cytop1asm, it is difficu1t to see why surplus ACh cannot be mobi1ized for re1ease by nerve impulses. The simp1est exp1anation is that depot ACh is synthesized at its site of storage, that is, in the vesic1es. A1ternative1y, if ChAc is a soluble cytop1asmic enzyme as Fonnum's (1966, 1967) resu1ts suggest, then a mechanism must exist for 84 packaging transmitter into vesicles which differs from the simple release of ACh from ChAc into the cytoplasm. It is possible that ACh which is to be used for transmission can only be packaged into vesicles at the moment of its synthesis, while ACh molecules liberated from ChAc after synthesi~ and preserved in the eytoplasm by an anti-ChE agent, can no longer become available. ChAc itself may play an integral part in placing ACh into vesicles. Further speculation about the site of synthesis and storage of ACh is unwarranted on the basis of the present experiments. The demonstration in the present work that surplus ACh does not mix with releasable ACh helps to clarify the interpretation of experiments by Collier and MacIntosh (1969). These authors labelled the ganglion's normal ACh store by perfusing an active ganglion with 3H-Ch-Locke in the absence of an anti-ChE; subsequent nerve stimulation, when perfusion medium contained eserine and unlabelled Ch, released ACh that had a specifie activity only 40% of that of the ganglion's ACh at the end of the loading perfusion. This and other experiments lead to the suggestion that theze was a pre ferential release of newly-synthesized transmitter, but the results were not unequivocal, and Collier and MacIntosh could not eliminate another possibility. In their experiments, Collier and MacIntosh tested release into eserine-Ch-Locke and the ganglia were therefore accumulating surplus ACh; if this surplus ACh mixed with releasable ACh, it would have diluted the specific activity and this cou1d account, at least partly, for their results. The experiments in this thesis demonstrate that surplus ACh and depot ACh do not mix, and therefore the only explanation for the resu1ts of Collier and MacIntosh (1969) is that there is indeed a preferentia1 release of newly-formed transmitter. Collier (l969) has reeently confirmed this 85 by repeating the earlier experiments but using neostigmine rather than eserine as the anti-ChE; he perfused for only 25 min with neostigmine- Ch-Locke, and during this period little surplus ACh formed; there was still a difference between the specific activity of released ACh and that in the ganglion. The preferential release of newly-synthesized transmitter has also been demonstrated recently at the mammalian neuromuscular junction (Potter, 1970). The failure of surplus ACh to enter the releasable ACh store in the present experiments is reminiscent of Marchbanks' (1968a) experiments on isolated synaptosomes that were prepared from brain tissue. Marchbanks 14 found that incubating synaptosomes with C-ACh labelled the cytoplasmic ACh but did not label ACh in vesicles. Fur thermore, the same author (Mar chbanks, 1969) found that synaptosomes exposed to l4C_Ch synthesized labelled ACh, but only cytoplasmic ACh and not vesicular ACh was labelled. Although the similarities between the present experiments and those of Marchbanks are tempting, other workers appear to have demonstrated the uptake of ACh into isolated vesicles (Gu th , 1969), as weIl as the labelling of vesicular ACh after incubating isolated synaptosomes with radioactive Ch (Potter, 1968). The reasons for these discrepancies between different workers using brain tissue is not obvious. D. Release of Surplus Acetylcholine by ~ High K+ depolarizes neuronal membranes and releases depot ACh from the superior cervical ganglion (Brown and Feldberg, 1936a) and the present experiments show that raised K+ also releases surplus ACh. It is possible that surplus ACh may be held within the nerve terminal 86 cytoplasm as a result of the existing membrane potential; a decrease in transmembrane potential in high K+ would then release surplus ACh. Several experiments of other workers are consistent with this idea. Superior cervical ganglia perfused with 40 mM KCl (7 x normal) appear not to accumula te surplus ACh (Birks, 1963); the synthesis of ACh by brain slices O1ann et al., 1939) or by brain synaptosomes (Marchbanks, 1969) is reduced in the presence of high K+ and ACh synthesis by these preparations might be analogous to surplus ACh formation by ganglia. The uptake of ACh into brain slices is inhibited by high concentrations of K+ (Polak, 1969; Liang and Quastel, 1969); much of the ACh transported by brain slices is retained in the nerve ending cytoplasm (Schuberth and Sundwall, 1968) and may therefore be analogous to surplus ACh in superior cervical ganglia. High K+ has been used to mimic the action of nerve stimulation in inducing the release of likely transmitter substances from brain slices: e.g., ACh (Mann et al., 1939; Polak and Meeuws, 1966; Sharkawi and Schulman, 1969), GABA (Machiyama, Balazs and Richter, 1967; Srinivasan, Neal and Mitchell, 1969), noradrenaline (Baldessarini and Kopin, 1967), glutamic acid (Katz, Chase and Kopin, 1969) and serotonin (Chase, Breese and Kopin, 1967). The experiments described in this thesis suggest that release of potential neurotransmitter substances by high K+ should be interpreted cautiously, for high K+ -Locke effectively released surplus ACh but nerve stimulation did note E. Réleaséof Surplus Acetylcholine by Acetylcholine or by Carbachol Koelle (1961, 1962) suggested that the small amount of ACh released 87 by nerve stimulation is not enough to propagate an action potential across a cholinergic synapse, but may înduce the release of sufficient ACh from presynaptic terminaIs to effect transmission. It is now established that ACh can depolarize non-myelinated nerves (Armett and Ritchie, 1960), in cluding preganglionic sympathetic nerve endings (Koketsu and Nishi, 1968) and it can induce antidromic firing at certain nerve terminaIs (Masland and Wigton, 1940; Riker, Roberts, Standaert and Fujimori, 1957; Randi~ and Straughan, 1964). VoIle and Koelle (1961) showed that the sensitivity of superior cervical gang lia to carbachol decreased when ganglia had been chronically decentralized, as if a part of its action on normal ganglia was due to its ability to release ACh. Although this difference in the sensitivity of normal and decentralized ganglia to carbachol has been questioned (Brimblecombe and Sutton, 1968; Brown, 1969), McKinstry et al., (1963) and McKinstry and Koelle (1967) clearly demonstrated that injected carbachol can induce the release of ACh from the perfused superior cervical ganglion of the cat. Collier, Vickerson and Varma (1969) labelled the ganglion's depot store and showed that injected or perfused ACh was ineffective in releasing this labelled ACh, although nerve stimulation released labelled ACh as expected; carbachol has also been shown unable to release depot ACh (Collier and Katz, unpublished observations). In the experiments of McKinstry and her co-workers which showed ACh release by carbachol, the ganglia were perfused with eserine and Ch and would therefore synthesize and store surplus ACh. It seemed possible that the whole of the ACh released by carbachol in those experiments was surplus ACh. The present experiments confirm that injected carbachol 88 releases surplus ACh, and demonstrate that ACh itself shares this effect. Surplus ACh was released more easily by injected carbachol, than by perfused carbachol, and the reason for this difference is not clear. In most experiments the effect of perfused ACh on surplus ACh release was transient and this was probably due to desensitization. The release of surplus ACh by ACh or by carbachol was not likely the result of presynaptic depolarization, because the concentration of ACh necessary for demonstrating the release of surplus ACh was below that necessary to depolarize presynaptic nerve terminaIs in frog and rat sympathetic ganglia ~~oketsu and Nishi, 1968). Cation exchange of ACh or carbachol for intracellular ACh was suggested by McKinstry and Koelle (1967) to be the mechanism for thê release of surplus ACh by these drugs, and this may be the most likely explanation. However, the possibility cannot be wholly eliminated that the release of surplus ACh by these drugs was secondary to K+ release as the result of their depolarizing action on the postsynaptic membrane (Katz, 1962); this is unlikely since ACh released by nerve stimulation failed to release surplus ACh, but stimulated postganglionic cells. If exogenous ACh exchanges with surplus ACh, it must be by an uptake mechanism ~nlike that in brain for that process is blocked by eserine (Polak and Meeuws, 1966; Schuberth and Sundwall, 1967; Polak, 1969; Liang and Quastel, 1969) and the present experiments demonstrated the release of surplus ACh by ACh in either eserine or DFP. The failure of nerve stimulation to release surplus ACh indicates that the concentration of released ACh at the site of storage of surplus -6 ACh is below 0.5 ~g/ml (2.8 x 10 M) which was the lowest concentration of exogenous ACh tested for this effect. Although the concentration of ACh 89 in the synaptic gap during nerve stimulation is not known with certainty, it has been estimated to be greater than 0.5 ~g/m1 (see e.g. Nishi, Soeda and Koketsu, 1967; Collier and MacIntosh, 1969). This indicates that surplus ACh is not localized at the ACh release sites; it might be distributed throughout nerve ending cytoplasm. The diffusion of neuronally released ACh (Ogston, 1955) would prevent the accumulation of effective ACh concentrations needed to release surplus ACh. If surplus ACh is stored in presynaptic cytoplasm distant from transmitter release sites, it is not surprising that the surplus ACh released by carbacho1 did not contribute to the drug's ganglion-stimulant effects. The release of surplus ACh by ACh or by carbachol may be analogous to the action of the indirect acting sympathomimetics in inducing the release of noradrenaline from adrenergic nerve terminaIs (Burn, 1932, reviewed by Trendelenburg, 1963). 90 v. SUMMARY 91 1. The formation and release of surplus ACh by the cat's superior cervical ganglion was studied using the standard techniques of ganglion perfusion. Surplus ACh is the_ extra ACh which accumulates in resting ganglia provided with a supply of Ch and exposed to an anti-ChE agent. 2. Surplus ACh formed in Ch-Locke solution containing either eserine, neostigmine or ambenonium, but its accumulation was delayed when neostigmine or ambenonium was the anti--ChE. The release of ACh by nerve stimulation was the same in either eserine, neostigmine or ambenonium-Ch-Locke; this shows that functional AChE was inhibited equally by each of these agents. The delayed formation of surplus ACh in the presence of neostigmine or ambenonium must therefore have been dependent upon the inactivation of another AChE pool. These results were discussed in relation to the histochemical localization of AChE. 3. Surplus ACh did not accumulate in chronical1y (1 week) decentra1ized ganglia. This supports the idea that surplus ACh is synthesized in the presynaptic fibres and not in the postsynaptic adrenergic cell body. 4. Surplus ACh was labe1led by perfusing the resting ganglion for 60 min with eserine-3H-Ch-Locke; approximate1y 50% of the surplus pool was 1abe1led. 5. Nerve stimulation failed to re1ease radioactivity from gang1ia whose surplus ACh had been label1ed. Re1ease was tested during perfusion with either eserine-Ch-Locke or eserine-HC-3-(Ch free)-Locke; nerve stimulation released only unlabe11ed ACh. This suggests that surplus ACh does not mix with the releasab1e depot pool, nor can surplus ACh serve as a source of 92 transmitter ACh. It also suggests that only "vesicular" ACh is available for release by nerve impulses. The relevance of these results to ACh synthe sis and storage was discussed. 6. The turnover of surplus ACh when its full complement had formed was calculated as 1/3 its rate of formation in a fresh preparation. + 7. 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