Beta Adrenergic Receptors of Guinea-Pig Atria and Trachea: the Use of Isometric-Activity-Differences of Agonists and Antagonists to Characterize Pharmacological

Home , Pronethalol

BUCKNER II, Carl Kenneth, 1941- BETA. ADRENERGIC RECEPTORS OF GUINEA-PIG STRIA AND TRACHEA: THE USE OF ISOMERIC- ACTIVITY-DIFFERENCES OF AGONISTS AND ANTAGONISTS TO CHARACTERIZE PHARMACOLOGICAL RECEPTORS.

The Ohio State University, Ph.D., 1970 Pharmacology University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED BETA ADRENERGIC RECEPTORS OF GUINEA-PIG ATRIA AND TRACHEA;

THE USE OF ISOMERIC-ACTIVITY-DIFFERENCES OF AGONISTS

AND ANTAGONISTS TO CHARACTERIZE

PHARMACOLOGICAL RECEPTORS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

-.1.i • • Carl Kenneth Buckner^ B.S., M.S

The Ohio State University

1970

Approved by

Adviser College of Pharmacy ACKNOWLEDGMENTS

I wish to express my sincere appreciation to

DRS. POP AT N. PATIL AND ARTHUR TYE for their instruction, guidance and support in class and research activities

MY WIFE, JEAN for her encouragement, endurance and understanding

MY MOTHER for her inspiration to achieve academic success

MY GRANDPARENTS for their invaluable support

DEDICATED TO MY BROTHER Robert Niel Buckner (1948-1970) VITA

November 27, 19 41 Born - Chandler, Oklahoma

1965 ...... B.S. in Pharm., University of Oklahoma, Norman, Oklahoma

1965-1968 ...... Teaching Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio

1968 ...... M.S., The Ohio State University, Columbus, Ohio

196 8-1970 ..... Research Assistant, College of Pharmacy, The Ohio State University, Columbus, Ohio

PUBLICATIONS

"Steric Aspects of Adrenergic Drugs. XII. Some Peripheral Effects of (± )•-eryth.ro- and (±)-threo-Methylphenidate.11 J. Pharmacol. Exp. fHer. 166: 308-3l9, 1969.

"Beta Receptors in Guinea-Pig Atria (A) and Trachea (T)." Pharmacologist 12: 285, 1970.

FIELDS OF STUDY

Major Field: Pharmacology

Structure-Activity Relationships of Adrenergic Drugs

Autonomic Nervous System

Professors Popat N. Patil and Arthur Tye (Retired)

iii TABLE OP CONTENTS

Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iii

LIST OF TABLES ...... Vi

LIST OF ILLUSTRATIONS...... viii

Chapter

I. INTRODUCTION

Adrenergic Receptors; Some Historical Concepts ...... 1

Classification of alpha and beta Adrenergic Receptors ...... 4

Subclassifications of beta Adrenergic Recep tors ...... 9

Statement of the P r o b l e m ...... 30

II. METHODS AND MATERIALS

General Considerations ...... 35

Isolated Right Atria ...... 36

Isolated Trachea Strips ...... 36

Use of Optical Isomers of Agonists ...... 37

Use of Optical Isomers of Antagonists .... 40

Drugs and Solutions ...... 43

III. RESULTS

Isomeric-activity-differences of beta Receptor A g o n i s t s ...... 46 Chapter Page

Influence of Tropolone on Isomeric-activity- differences of Agonists ...... 55

Influence of Theophylline on the Isomeric- . activity-difference of Isoproterenol in Guinea-pig Atria ...... 68

Isomeric-activity-differencesof beta Recep tor Antagonists ...... 71

Combinations of Isomers of beta Receptor Antagonists ...... 86

Rate of Onset of beta Receptor Blockade . . 87

IV. DISCUSSION ...... 99

V. SUMMARY AND CONCLUSIONS ...... 116

APPENDIXES ...... 119

BIBLIOGRAPHY ...... 124

V LIST OF TABLES

Table Page

1. Chemical Structures of Some beta Adrenergic Receptor Agonists ...... 21

2. Chemical Structures of Some beta Adrenergic Receptor Antagonists ...... 23

3. Changes in Sensitivity of Guinea-pig Tissues Obtained from Separate Control Experi- men ts ...... 44

4. Effects of Optical Isomers of beta Receptor Agonists on Guinea-pig Atria and Trachea . 53

5. Effects of Optical Isomers of beta Receptor Agonists Obtained from the First Curves of Each Experiment in Guinea-pig Atria and Trachea ...... 56

6. Effects of the Isomers of Catecholamines in the Absence and Presence of Tropolone . . . 67

7. Effects of beta Receptor Antagonists on (-)- Isoproterenol-induced Increases in Guinea- pig Atrial Rate ...... 78

8. Effects of beta Receptor Antagonists on (-)- Isoproterenol-induced Relaxation of Guinea- pig Tracheal Strips ...... 79

9. Effects of Combinations of Optical Isomers of beta Receptor Antagonists on Dose Ratios Obtained on Guinea-pig Atria ...... 90

10. Effects of Time of Antagonist Incubation on Blocking Potency of Isomers of Alprenolol in Guinea-pig Atria and Trachea ...... 95

11. Percent of Log (Dose Ratio) Values at Dif ferent Times of Incubation of Isomers of Alprenolol in Guinea-pig Atria and Trachea . 98

vi Table Page

12. Formula Weights and Sources of Chemicals used in the Present Study ...... 120

13. Effects of (-)-Alprenolol, 10”7 M, at Various Time Intervals of Contact with Guinea-pig Atria and Trachea ...... 121

14. Effects of (+)-Alprenolol, 10“5 m , at Various Time Intervals of Contact with Guinea-pig Atria and Trachea ...... 122

15. Effects of (-)-Alprenolol, 10"^ M, at Various Time Intervals of Contact with Guinea-pig Atria and Trachea ...... 123

vii LIST OF ILLUSTRATIONS

Figure Page

1. Log-Dose-Response Curves for (-)- and (+)- Isoproterenol Obtained from Atria and Trachea taken from Normal and Reserpine-Pretreated Guinea-pigs ...... 48

2. Log-Dose-Response Curves for (-)- and (+)- Norepinephrine Obtained from Atria and Trachea taken from Normal and Reserpine- Pretreated Guinea-pigs ...... 50

3. Log-Dose-Response Curves for (-)- and (+)- Epinephrine Obtained from Atria and Trachea taken from Normal and Reserpine-Pretreated Guinea-pigs...... 52

4. Log-Dose-Response Curves for (-) Forms of Isoproterenol, Norepinephrine and Epine phrine Obtained from Atria and Trachea taken from Reserpine-Pretreated Guinea-pigs .... 58

5. Log-Dose-Response Curves for (-)- and (+)- Isoproterenol Obtained with and without Tropolone from Trachea taken from Normal Guinea-pigs...... 60

6. Log-Dose-Response Curves for (-)- and (+)- Isoproterenol Obtained with and without Tropolone from Atria taken from Normal Guinea-pigs ...... 62

7. Log-Dose-Response Curves for (-)- and (+)- Norepinephrine Obtained with and without Tropolone from Atria taken from Reserpine- Pretreated Animals ...... 64

8. Log-Dose-Response Curves for (-)- and (+)- Epinephrine Obtained with and without Tropolone from Atria taken from Normal Animals ...... 66

viii Figure Page

9. Log-Dose-Response Curves for (-)- and (+)- Isoproterenol Obtained with and without Tropolone, 3 X 10"5 m , and/or Theophylline, 1 0 M, from Atria taken from Normal Guinea-pigs ...... 70

10. Plots of Log (Dose Ratio - 1) vs. Molar Concentration of (-)- and (+)-Alprenolol Obtained from Guinea-pig Atria and Trachea . 73

11. Plots of Log (Dose Ratio - 1) vs. Molar Concentration of (-)- and (+)-Sotalol Obtained from Guinea-pig Atria and Trachea . 75

12. Plots of Log (Dose Ratio - 1) vs. Molar Concentration of (-)- and (+)-INPEA Obtained from Guinea-pig Atria and Trachea . 77

13. Plots of Log (Dose Ratio - 1) vjs. Molar Concentration of (±)-Practolol Obtained from Guinea-pig Atria and Trachea ...... 82

14. Plots of Log (Dose Ratio - 1) vs. Molar Concentration of (-)-Sotalol Obtained from Guinea-pig Atria and Trachea ...... 84

15. Log-Dose-Response Curves for (-)-Isoproterenol from Guinea-pig Atria in the Absence and Presence of (-) -, (+) - and (-)- and (+)- A l p r e n o l o l ...... 89

16. Plots of Log (Dose Ratio) vs. Time of Incuba tion with Equiactive Concentrations of the Isomers of Alprenolol from Guinea-pig Atria and Trachea ...... 94

17. Plots of Percent of Log (Dose Ratio) at 120 min vs. Time of Incubation with Isomers of Alprenolol from Guinea-pig Atria and Trachea . 97

18. Log-Dose-Response Curves for (+)-Isoproterenol and desoxy-Isoproterenol Obtained from Atria taken from Normal Guinea-pigs ...... 109

ix CHAPTER I

INTRODUCTION

Adrenergic receptors: some historical concepts

In his comprehensive study of the action of epine phrine, Elliott (1905) stated:

It is the peculiarity of adrenalin to cause sharp contraction in the one (tissue) and relaxation in the other.

He considered that these actions were produced through some substance which had developed from the muscle result ing from its innervation by sympathetic nerve fibers, but was not an intrinsic part of the muscle. The response observed would then depend upon the nature of the "myo neural junction". His professor, N. J. Langley, did not adopt these views completely since the response to epinephrine did not degenerate along with the sympathetic nerves after their section (Langley, 1905). Langley felt that the characteristic response was produced when epine phrine interacted with some constituent of the cell itself and concluded:

...a cell may make motor or inhibitory receptive substances or both, and that the effect of a nervous impulse depends upon the proporation of the two kinds of receptive substance which is affected by the impulse.

In his classical study of ergot alkaloids, Sir Henry

Dale (1906), confirming observations that the effects of 2 epinephrine could clearly be separated into two types, excitatory and inhibitory, summarized his data by stating:

... ergot contains a principle which has a para lytic action on the motor elements of that myo- trophic structure or substance which is excited by adrenaline and by impulses in fibers of the true sympathetic system; the inhibitor elements of the same being relatively or absolutely un affected.

Thus were made the first steps toward characterizing sympathomimetic responses in terms of the "receptive substances" mentioned previously by Langley.

However, because of the resemblances noted by Elliott

(1905) and Dixon and Hamill (1909), the inference was drawn that the substance liberated by sympathetic nerve impulses was epinephrine. Certain discrepancies between effects of intravenous epinephrine and sympathetic nerve stimulation were eventually noted by several workers. In

1910, Barger and Dale suggested that the effects of nor epinephrine corresponded more closely to effects of sym pathetic nerves than did epinephrine. Their statement went unpursued for several years.

The first significant conflict came in 19 33 when

Cannon and Rosenblueth apparently confirmed that sympathetic nerve stimulation resulted in the release of some factor other than epinephrine. They hypothesized that there were actually two different transmitters liberated from sympathe tic nerve terminals, sympathin E (excitatory) and sympathin

I (inhibitory). Thus, the responses observed would be dependent upon the relative predominance of one over the other and this would be determined by the tissue under investigation. It was further supposed that substances

E and I corresponded to Langley's "receptive substances."

In a study of the importance of various groups on the epinephrine molecule, Youmans et. al_. (19 39), using seven anologs of the parent molecule, concluded:

It is clear that no consistent relationship exists between the smooth-muscle-relaxing (intestine) and the smooth-muscle-contracting (nictitating membrane) properties of these amines. Such a result is in accord with the postulation of at least two types of smooth muscle with regard to 'receptive mechanisms.'

Their results were thus opposed to the views of Cannon and

Rosenblueth and supported the presence of Langley's

"receptive substances", one excitatory and one inhibitory.

With the aid of N-alkyl substituted catecholamines,

Lands et al. (19 47) proposed that there were definite structural requirements for depressor effects as compared with those necessary for pressor effects. They felt that these derivatives, among which was N-isopropylarterenol

(isoproterenol), should be designated as "sympathin I- mimetic agents" since their major effects were to mimic the inhibitory actions of epinephrine. The main exception to general inhibition was their marked stimulating effects on the heart. This action was explained as resulting from liberation of sympathin E. The controversy over the sympathetic nerve substance was subsequently dissolved by the work of Euler (1946, 1948)

and reviewed by him in 19 56. Thus, the results of Cannon

and Rosenblueth could largely be explained on the basis

of norepinephrine being the transmitter substance. However,

the matter of inhibitory ws. excitatory sympathomimetic

responses was not entirely solved.

Classification of alpha and beta adrenergic receptors

In 19 48, Ahlquist, selecting six closely related

catecholamines, reported orders of potencies on a wide

variety of tissues and responses. Importantly, his studies

included results from the heart and intestinal smooth muscle

of several species. Among the several tissues, there were

clearly two distinct orders of potencies for the six

sympathomimetic agents. In some tissues, isoproterenol

was the most potent, while in the others, it was least

potent. These results suggested to Ahlquist, as it had to

previous workers, that there are two different types of

sympathomimetic receptor. It was clear, however, that

they could not be accurately classified as excitatory or

inhibitory because these receptors produced either action

depending upon where they were found. Ahlquist therefore

suggested that the sympathomimetic (or adrenotropic)

receptors be classified as either alpha or. beta depending

upon their relative responsiveness to a series of sympatho

mimetic amines. Activation of the alpha receptor would result primarily in excitatory activities except in the intestine where it produced inhibition. Conversely, activation of the beta receptor would be associated with most of the inhibitory functions, the one exception being excitation of myocardial tissue.

It appears that Ahlquist's classification went largely unnoticed or ignored at first. Usually, the postulation of separate receptors requires specific antagonists. In

19 48, the only available antagonists to sympathomimetic responses were those which blocked their excitatory actions, excluding those of the heart. For example, ergot alkaloids had been characterized by Dale in 1906. Nickerson (1949) reviewing the development of adrenergic antagonists, summarized:

It is generally agreed that no adrenergic blocking agent specifically inhibits the chronotropic and inotropic responses of the mammalian heart to adrenergic stimuli.

It seemed clear that the beta receptors of the heart, if not the same as those responsible for inhibition, were equally not the same as those producing excitation in smooth muscle.

After studying the optical isomers of norepinephrine and relating their actions to those of epinephrine and isoproterenol, Luduena et al. (19 49) concluded:

The affinity for the same cellular mechanism would depend on the structural feature which they (amines) have in common while the type of amine group would be responsible for the direction and degree in which this mechanism is influenced. The implication was that since all of the amines studied

could produce a combination of stimulation and inhibition

depending upon the measured response, the results could be

explained by different substituents on the drug molecule

rather than different receptive mechanisms in the tissues.

However, if that were true, the available adrenergic an

tagonists should have blocked, to some extent, inhibitory

as well as excitatory effects.

Concerned about Ahlquist's classification, particularly

with regard to the placement of heart responses in the same

category as inhibitory responses, Lands (1952) investigated

a large series of sympathomimetic agents in an attempt to

find optimal structural requirements for the proposed

receptor systems. All of the amines increased both rate

and amplitude of the heart beat. He concluded that excita

tion was a result of interaction with the "Ac" receptor,

inhibition occurring through the "Ar" receptor. The

receptor of the heart was said to be undifferentiated and

categorized as "Acr". It could be stimulated by sub

stances with an affinity for either "Ac" or "Ar" receptors.

However, the "Acr" receptor could not be blocked by avail

able antagonists.

Ten years after the classification of alpha and beta

receptors, in 1958, Powell and Slater reported that the

dichloro derivative of isoproterenol was able to selectively

antagonize inhibitory responses to sympathomimetic amines. In the same year, Moran and Perkins (1958) showed that this compound could also block sympathetic cardiac responses.

It thus seemed that sympathetic smooth muscle inhibition and cardiac excitation were both produced through an identi cal receptor. These observations substantiated the concept of alpha and beta receptors and began a new era of extensive investigations into adrenergic receptive mechanisms.

There was, however, at least one point of controversy which had not been resolved. On the basis of observed orders of potencies, Ahlquist had classified inhibition of intestinal smooth muscle as alpha. As discussed by

Nickerson (19 49), dibenamine, an antagonist of most excita tory responses, was unable to effectively block the actions of catecholamines in rabbit intestine. In reviewing adrenergic receptors, Furchgott (1959) reported that low concentrations of dihydroergotamine could antagonize effects of similarly low concentrations of epinephrine, but the antagonism was easily overcome by a 20 fold higher concen tration of the latter. In addition, the inhibitory action of the higher concentration of epinephrine could not be blocked by a 20 fold higher concentration of dihydroergota mine. He also confirmed that neither dibenamine nor dichloroisoproterenol could block this intestinal response.

Furchgott then proposed that the receptor involved in intestinal inhibition be classified as delta.

The question of the intestinal receptor was settled in the same year by Ahlquist and Levy (19 59) using agonists and antagonists which were specific for either type of adrenergic receptor. Intestinal inhibition produced by phenylephrine, an agonist with greater affinity for alpha receptors, was effectively antagonized by the alpha blocking drugs, dibozane, dibenamine and phenoxybenzamine. Similarly, inhibition produced by isoproterenol was blocked by its dichloro derivative. In addition, a combination of both types of antagonist could block responses to epinephrine, an agonist with good affinity for both alpha and beta receptors. The obvious conclusion was that both alpha and beta receptors are present in intestinal smooth muscle and stimulation of either results in relaxation or inhibition.

This observation has been confirmed by Van Rossum and

Mujic (1965).

By 1964, the beta adrenergic blocking properties of dichloroisoproterenol had been thoroughly investigated and two more potent antagonists were introduced; pronethalol

(Black and Stephenson, 1962) and propranolol (Black et al.,

19 6 4). The concept of alpha and beta adrenergic receptors was internationally accepted. It was a simple, convenient way to predict and classify adrenergic drug responses.

Compounds which blocked alpha receptor responses were termed alpha adrenergic antagonists and those which blocked beta receptor responses were beta adrenergic antagonists (Moran and Perkins, 1958). It was also recognized that the adrenergic blocking agents could distinguish between the two receptors more precisely than most agonists (Ahlquist and Levy, 1959). Pharmacologically, there were two ways to characterize adrenergic receptors: 1) specific activa tion by agonists (relative potencies) and 2) specific blockade by antagonists. With these criteria, it was considered that the heart contained only beta receptors, while the intestine contained both types of adrenergic receptor. Most other tissues responded to alpha and beta receptor activation by contracting or relaxing, respectively

Subclassifications of beta adrenergic receptors

Lands and Brown (196 4) studied a large number of structurally related catecholamines on three responses mediated via beta adrenergic receptors: bronchodilation, positive chronotropic and positive inotropic responses of cardiac tissue. They noted that the molecular require ments for effecting each parameter was specific and differed considerably from the others. Just as Youmans et al.(1939) had separated excitatory from inhibitory and Ahlquist (1948) had classified alpha and beta receptors, Lands and Brown

(196 4) proposed that responses considered to be mediated through beta receptors could be further classified accor ding to orders of potencies of agonists. However, the available antagonists could block beta receptor responses in all tissues which contained these sites in the same dose range. Thus, results from antagonism suggested that all beta receptors are identical.

By this time, the receptors involved in mediating the 10 metabolic responses (increase in blood glucose and free fatty acids) to catecholamines were suspected of being beta since these effects were antagonized by dichloroisoproterenol

(Mayer et al,, 1961). The metabolic responses to epine phrine were also antagonized by two new agents, methoxamine and its N-isopropyl analog (Burns et aJ., 1964).

Levy (1964) studied the effects of N-isopropylmethoxamine and methoxamine on beta adrenergic responses in anes thetized dogs. Neither drug was able to block the vaso depressor, positive inotropic, positive chronotropic or

intestinal inhibitory responses to isoproterenol. All of

these responses were effectively antagonized by dichloro-

isoprotere.nol and pronethalol. In isolated rat uterus,

N-isopropylmethoxamine and methoxamine blocked catechola mine-induced inhibition in a manner similar to the other beta receptor antagonists. Levy considered two possible explanations for these results. Methoxamine and its

analog could be considered as specific beta receptor

antagonists or the beta receptors in the rat uterus were different from those in other tissues.

VanDeripe et. al. (1964) had reported results from

other a-methylated blocking agents and observed that a- methyldichloroisoproterenol and dichloroisoproterenol were equally active in antagonizing the vasodilation

produced by isoproterenol. However, dichloroisoproterenol was 15 times more active than its a-methyl derivative in 11 antagonizing the positive inotropic actions of isopro terenol. In discussing these data, Moran (1966) concluded that it was not possible to decide whether there are different beta receptors in heart vs. peripheral vascula ture or whether the a-methyl analogs act at some site distal to the receptor in antagonizing the vascular response. In view of the slight structural modification of the blockers, he felt that the former explanation was more likely to be true and suggested that beta receptors may be better classified according to the tissues in which they are found.

Another a-methylated antagonist, N-tertiary-butylmethoxamine (butoxamine) was studied by Levy (1966a).

This substance was also found to differ from dichloroiso proterenol in that it did not alter, in anesthetized dogs, intestinal inhibition, positive chronotropic or positive inotropic responses produced by isoproterenol. Like N- isopropylmethoxamine, methoxamine and dichloroisoproterenol, butoxamine was able to antagonize beta receptors of rat uterus. Butoxamine had also been shown to block metabolic responses to catecholamines (Burns and Lemberger, 1965).

From these data, it appeared that butoxamine had the same profile of activity as did methoxamine. There was, however, one notable difference. Butoxamine was able to antagonize vasodilator responses to isoproterenol in the femoral vascular bed of the dog, an action not displayed by methoxamine. Furthermore, other antagonists with close 12 structural resemblance to butoxamine, dimethyl isopropylmethoxamine (Levy, 1966b) and 1135/25 (Levy, 1967a), had the same spectrum of blocking activity as did butoxamine and a-methyldichloroisoproterenol (VanDeripe et al., 1964).

At this point, it was interesting that all of the compounds which could block some, but not all, beta receptor responses had an a-methyl group. These drugs were considered "selective" and, on this basis, it was considered by many that beta receptors were not homo geneous in all tissues. The so-called "selectivity" might also be explained by the slight structural differ ences between a-methylated and non-a-methylated deriva tives (Levy, 1966b). The principle pharmacological differences seemed to be in their abilities to antagonize sympathomimetic responses in the heart and peripheral vasculature. None of the a-methyl compounds could effectively block the beta receptors of the heart, but some had good affinity for these receptors in the vascular beds. In addition, all of the known beta receptor antagonists were able to antagonize metabolic and rat uterine responses to catecholamines in a similar manner.

The non-a-methylated derivatives were considered as

"classical" beta receptor blocking agents since they could block beta receptor responses in all tissues in the same dose range (Wilkenfeld and Levy, 1968).

Structural variations of beta receptor agonists had 13 not gone unstudied. As mentioned previously, other investigators, notably Lands and his colleagues, were actively engaged in determining structural requirements for beta receptor activation.

Among the several compounds studied by Lands and

Brown (1964) were some a-methyl and a-ethyl derivatives.

Such structural modifications reduced activity in the heart and lung when compared to the parent non-substituted compounds. However, the same degree of reduction did not occur in both tissues. For example, a-ethyliso- proterenol was considerably less potent than isopro terenol in producing a positive chronotropic response in perfused rabbit heart, but was one-half as active as a bronchodilator in guinea-pig perfused lung. It was also clear that norepinephrine was relatively more active in the heart than lung. The same was true of a-methylnorepinephrine, but it was equally active to norepine phrine in lung and about one-half as active in heart.

Holtz and Palm (1967) also investigated a-methylated amines using anesthetized cats and isolated guinea-pig atria. They demonstrated that a-methylepinephrine and a-methylnorepinephrine had higher affinities for vascular beta receptors than their non-a-methylated compounds.

Norepinephrine and a-methylnorepinephrine were equally active in the guinea-pig atria, but epinephrine was 4 times more potent than a-methylepinephrine on this tissue. 14

Furthermore, after blocking alpha receptors with phenoxybenzamine, dopamine (desoxy-norepinephrine) had greater vasodepressor effects than norepinephrine. However, this response to dopamine was not abolished by pronethalol while the vasodepressor responses to a-methyldopamine were reversed. It was concluded that dopamine becomes a vascular beta receptor agonist only after a-methylation.

In the heart, a-methyldopamine was on l/6th as active as norepinephrine. Dopamine, with predominantly indirect

(norepinephrine releasing) actions in this tissue, was

50 times less potent than norepinephrine. Thus, dopamine- induced beta receptor activation in guinea-pig atria also becomes significant only after its a-methylation.

The in vitro studies of Van Rossum and Mujic (1965) gave corresponding results from rabbit intestine. In this tissue, after blockade of alpha receptors, relaxation produced by a-methylnorepinephrine became more pronounced than that produced by (-)-norepinephrine.

Generally, evidence from agonists and antagonists indicated that the influence of a-methylation was to considerably reduce the apparent affinities of these drugs I for cardiac beta receptors. Addition of an a-methyl group usually increased vascular and intestinal beta receptor agonist potencies and slightly reduced bronchodilator activity.

N-alkyl substituted derivatives of catecholamines

also displayed different profiles of action on the / 3-5 several responses. Thus, butoxamine was active in blocking beta receptors of rat uterus and femoral arteries of the dog, but relatively ineffective in antagonizing cardiac

and intestinal beta receptors (Levy, 1966a; Levy, 1967b).

Similarly, the N-tertiary-butyl analog of norepinephrine was reported to be more potent than isoproterenol, the N-

isopropyl derivative, in effecting brochodilation in guinea

pigs and vasodepression in anesthetized dogs, but less

active in producing positive inotropic and chronotropic

responses in isolated perfused rabbit hearts (Lands et al.,

1966).

Attempting to bring the several results in perspec

tive, Lands et al. (1967a, b) suggested that, depending

upon relative potencies of structurally varied catechola

mines , beta receptors be divided into 3“1 and 3-2 sub-

types . In accordance with their results and those pre

viously mentioned, beta receptors responsible for positive

inotropic and chronotropic responses of the rabbit heart,

rabbit small intestinal inhibition and lipolysis in rat

testicular adipose tissue were arbitrarily placed in the

3-1 category. Beta receptors of the rat uterus and dia

phragm, guinea-pig trachea and vascular system of the

anesthetized dog were classified as 3-2. The activities

of a large number of sympathomimetic amines were related

to the effects of isoproterenol and relative potencies on

several tissues were determined. Whenever the rank order 16 from one tissue correlated with the rank order from another, these tissues were considered to contain the same subclass of beta receptor.

On this basis, there was no doubt that different orders of potencies could be observed in different tissues, in vivo and in vitro. According to Lands et al. (1967a, b ) f this apparent "selectivity" was due to different receptor types. Could the observations be explained otherwise?

Furchgott (1967), in a critical appraisal of the use of orders of potencies of agonists to differentiate receptors, indicated that such studies could be valid only after applying proper experimental procedures to eliminate complicating factors. The most important criteria to be satisfied for ideal comparison are:

1) Each agonist must produce its effects through direct activation of the receptor system to be studied.

Any action dependent upon release of endogenous transmitter should be eliminated. For example, tyramine would not be an ideal agent since a major portion of its action is produced indirectly by liberation of norepinephrine from

adrenergic nerve terminals. Also, the indirect component of dopamine and other agents with mixed action (indirect

and direct) should be eliminated prior to testing their

potencies.

2) All of the effects observed from each agonist must be due to activation of only the receptor system to be 17 studied. It is well recognized that many sympathomimetic amines can activate both alpha and beta receptors directly.

Most tissues are known to contain both receptor types and responses elicited through them are not necessarily opposed.

In intestinal smooth muscle, activation of either alpha or beta receptors results in inhibition or relaxation. In most other tissues, however, effects mediated through

these receptors are antagonistic. Therefore, in order to

study one receptor, exclusive of the other, an appropriate

antagonist of the other must be applied. Otherwise, the

observed response will be the sum of the effects pro duced by activation of both receptor systems.

3) Factors which are responsible for decreasing (or

increasing) the concentration of each agonist in the

region of the receptors must be controlled. For agents

like norepinephrine and epinephrine, the most effective

removal process appears to be adrenergic neuronal membrane uptake (Iversen, 1967). Other agents may be taken up by this active process to varying degrees. The end result

is to alter the concentration at the receptors such that,

in many cases, the observed response is drastically

reduced. Therefore, in order to more accurately determine

the potencies of individual amines at their receptors, this

concentrating mechanism must be eliminated by sympathetic

denervation or addition of an inhibitor of uptake, eg.,

cocaine. After such procedures, other routes for disposal 18 of the amines may become more important, for example, metabolism by the enzyme catechol-o-methyltransferase

(COMT). An appropriate inhibitor of this enzyme must then be applied.

Obviously, all of these factors should be considered in determining the pharmacological activities of a series of sympathomimetic amines. However, Furchgott (1967) was the first to attempt to eliminate most of them. With the aid of only directly acting amines after blockade of alpha receptors with phentolamine and adrenergic neuronal membrane uptake with cocaine or phenoxybenzamine, he showed that relative beta receptor potencies could be considerably altered. Most notably, in guinea-pig and rabbit heart and guinea-pig duodenum there was a change in the rank order after cocaine such that norepinephrine, which had been less active than epinephrine, became more active. In some tissues, e £ . , rabbit aortic strips, there was almost no change in relative potencies of the agonists after drug treatments.

In order to further characterize beta receptors,

Furchgott (1967) utilized the competitive antagonist, pronethalol. Through currently accepted receptor theory, determination of the dissociation constants (KB) of the receptor-antagonist complex is a simple procedure, pro vided certain assumptions are valid. At the time responses are measured, antagonist, agonist and receptor molecules are assumed to be in equilibrium (steady state). It is 19 also assumed that equal responses are produced by equal receptor occupancies before and after the antagonist

(Arunlakshana and Schild, 19 59). In addition, increasing concentrations of antagonist should not alter its KB .

However, since the true concentration of antagonist in the region of the receptors is unknown, the measured KB value is only apparent.

As with the use of agonists, proper experimental conditions are necessary for a valid study of antagonists.

When many adrenergic agonists are used to determine dose ratios, the conditions mentioned previously must be satis fied. The ideal agonist for such experiments is one with low potency for other than the specific receptor. For example, isoproterenol is the agonist of choice for studying beta receptors. Not only does it have low potency for activating alpha receptors, it is not significantly taken up by adrenergic nerve terminals (Hertting, 1964). However, to prevent alpha receptor activation at high dose ratios, an appropriate antagonist should be applied. In addition, all of the antagonism produced by the beta receptor blocker should be due only to combination with that specific recep tor. Also, the tissue should be left in contact with the antagonist for a time sufficient to achieve equilibrium.

Arranging the tissues into three groups according to apparent KB values for pronethalol, as was done for rela tive potencies of agonists, Furchgott (1967) recognized 20 that both criteria gave complementary results. Further more, the classifications corresponded reasonably well with the 0-1 and 0-2 categories, excepting rabbit duo denum and stomach which were proposed to belong to a third subtype. Thus, guinea-pig tracheal and rabbit aortic beta receptors belonged together, but apart from beta receptors in atria from both species. Such classes were also substantiated by the in_ vivo observations of

Levy (1964; 1966a, b; 1967a, b) regarding the "selective" nature of beta receptor blockade exhibited by a-methylated antagonists.

Additional support for the beta receptor classifi cations was given by several investigators who recognized the possibilities of developing "selective" beta receptor drugs for clinical use.

All of the so-called "selective" beta receptor agonists subsequently developed were shown, through various techniques, to possess greater relative activities in re laxing tracheal smooth muscle as opposed to stimulation of the myocardial parameters. The more important and interes ting of these agonists are soterenol or MJ 1999 (Larsen et al. , 1967; Dung an' et al. , 1968), quinterenol (Scriabine et al., 1968), salbutamol or AH 3365 (Brittain et al., 1968;

Farmer and Levy, 1968; .Cullum et al., 1969) and terbutaline

(Bergman et al., 1969). Chemical structures of several beta receptor agonists are shown in Table 1. The compound 21

TABLE 1

Chemical Structures of Some Beta Adrenergic

Receptor Agonists.

Compound Structural Formula Oil Isoproterenol HO- -CH-CH2-NH-CH(CH3)2

ho"

0H Soterenol3 HO1O -CH-CH2-NH-CH(CH3)2 c h 3s o 2n h

Salbutamola h o -^jT\ V °Hc h -c h 2-n h -c (c h 3) 3

h o c h 2 HO ; j r \ ° H Orciprenaline (' XVCH-CH2-NH-CH (CH3) 2

HO OH // \ 1 Terbutalinea (' NN-CH-CH2-NH-C(CH3) 3

OH Quinterenola HO-^ \-CH-CH2-NH-CH (CH3) 2

u __ para-D imethy1- )CV/_\\.N(CH3)2 aminobenzaldehydea H '--

aCompound suggested to be a "selective" agonist. para-dimethylaminobenzaldehyde (DMAB) appears to possess specific and "selective" beta receptor agonist activity

(Ghouri and Shibata, 1970). The relatively weaker effects of this agent on isolated rabbit atria were apparently not abolished by reserpine-pretreatment and sotalol shifted its dose-response curves on rabbit ileum and aortic strips and guinea-pig taenia coli and trachea to the right in competi tive fashion. Phentolamine did not alter its activity on any tissue. DMAB was 72,000 times less active than isopro terenol in producing a positive chronotropic response and

122 times less potent in relaxing trachea. This spectrum of activity of DMAB is interesting in view of its lack of structural resemblance to the other beta receptor agonists.

(Table 1) .

In addition to the antagonists related to methoxamine, at least one important new beta receptor blocking agent, practolol (Barrett et al., 1968; Brick et al., 1968;

Dunlop and Shanks, 1968), has been shown to possess so- called "selectivity". As opposed to the "selective" agonists, practolol has greater potency in blocking the beta receptors of the heart. Chemical structures of several beta adrenergic receptor antagonists are shown in

Table 2.

It is interesting that none of the newer agonists or antagonists contain a substitution at the a-carbon atom. 23 TABLE 2

Chemical Structures of Some Beta Adrenergic

Receptor Antagonists.

Compound Structural Formula OH

Dichloroisoproterenol Cl-^ ^-CH-CH2 -NH-CH(CH3 ) 2

c {

tv. ; — v O H

Pronethalol ' V NVCH -CH 2 -NH-CH(CH3) 2

CH3q OH CH 3 i 1 Me thoxamine 3 Q ch-ch-nh2

soch3

CH30'-- OH CH 3 Butoxamine3 P V 6 h-CH-NH-C (CH3) 3

' OCH 3

H35/25 f\ ?H ?H3 CH3-V Vch-ch-nh-ch (CH3 ) 2

jT\ 9H H29/50 CH3-^,_// 'VcH-f CH- CH2 -NH-CH(CH3) 2

Propranolol (• n)-o-ch2 -ch-ch2 -nh-ch (CH3 ) 2

0------. OH

Practolol® ch 3 -c-nh-^ V o- ch2 -ch-ch2 -nh-ch (ch3) 2 aCompound suggested to be a "selective" antagonist. 24

To analyze in_ vivo blocking properties in anesthe tized dogs, Levy and Wilkcnfeld (1969) selected three drugs representing the known classes of beta receptor blocking agents. Each was tested for its ability to reduce vasodepressor, positive chronotropic and intestinal inhibitory responses to isoproterenol. H29/50 was representative of the "classical" beta receptor antagonists and, like propranolol and dichloroisoproterenol, blocked

all three beta receptor responses in the same dose range.

Only the vasodepressor responses were antagonized by

H35/25. Practolol, the newer drug, blocked only the positive chronotropic and intestinal inhibitory responses.

The observed selectivity was undeniable, however, the ex tent to which in vivo data can be used to determine receptor types is questionable. Thus, are the clearly different activities of these blockers due to different receptor types involved in the responses or are factors

affecting distribution and/or metabolism of these compounds

influencing their observed potencies? Clearly, in vitro studies on isolated tissues are expected to provide relatively better information.

Farmer et al.(1970) studied the relative potencies of

five beta receptor agonists on isolated atria and trachea

from the guinea pig. Some of the agonists were "selective"

in activating tracheal beta receptors (salbutamol, soterenol

and trimetoquinol) while others displayed no "selectivity" 25

(orciprenaline). All potencies were related to isopro terenol and since different orders were obtained in the two tissues, the results were interpreted as substantiating the classification proposed by Lands et al. (1967a, b) and

Furchgott (1967). However, the proper experimental con ditions defined by Furchgott were apparently not provided.

On this basis, the validity of these observations in differentiating the receptors involved is in doubt.

According to Furchgott (1967), measurement of apparent KQ values for receptor-antagonist complexes in several tissues should be useful in differentiating receptors and provide results consistent with those from relative potencies of agonists. As defined by Arunlakshana and SchiId (1959) , pA 2 = -log KB ; where pA 2 is the negative log of the concentration of antagonist required to reduce the response to a given concentration of agonist by one- half. After that concentration of antagonist, a double dose of agonist is required to produce a response equal to that before the antagonist. Whenever pA2 values for a given blocker differ between tissues, the receptors with which the blocker combines are assumed to differ in those tissues. The assumptions made for determining apparent KQ values are the same for determining pA2 values and in both instances, appropriate experimental conditions are necessary for accurate determinations (see above). In some cases, slope values for the regression lines in pA2 26 determinations are not equal to the theoretical value of

-1 (Rang and Ritter, 1969; Bristow et al., 1970; Levy and

Wilkenfeld, 1970). When this occurs, apparent KB values will vary with the concentration of antagonist and neither parameter is expected to accurately reflect the activity of the antagonist at its receptors. In many cases, such slopes are ignored and the calculated pA£ values taken as a basis for differentiating receptors (Farmer and Levy,

1970; Takagi and Takayanagi, 1970).

Attempting to differentiate beta receptors in several tissues, Farmer and Levy (1970) and Takagi and Takayanagi

(1970) determined pA 2 values for "classical" and "selective" antagonists in vitro. As expected from in vivo results, propranolol had similar pA 2 values in all tissues studied.

The "selective" antagonist, practolol, displayed at least

10 fold differences in pA^ values. Thus, the tissues could be divided into at least two groups corresponding closely to the 3-1 and 3-2 categories. However, slope values were not reported and proper experimental conditions were apparently not satisfied.

In a more carefully controlled study of beta receptors in several isolated tissues from the rabbit, Bristow et al.

(19 70) reported results with four beta receptor blocking agents. Apparent KQ values were calculated, but where slope values were less than -1, only apparent pA2 values could be determined. The results were considerably varied. 27

For some drugs there were only minor differences in activity between the tissues. Others displayed larger variations and some were active in one or more of the tissues, but not in the others. Furthermore, the results did not correspond to previous beta receptor classifica tions. For example, H35/25, previously considered to be

"selective" for 3“ 2 receptors, had similar apparent K q values in rabbit atria and aorta, classified as 3-1 and

3-2, respectively. In addition, propranolol had identical pA2 values in atria and trachea. The authors concluded that all four tissues contain different beta receptor types. However, the classifications could as easily be made on the basis of the individual drugs rather than the receptors involved.

A review of the classifications into which beta receptors of various tissues have been placed shows that there is no pharmacological criterion which accurately provides consistent results. Most of the data can be criticized on the basis of failure to provide proper experimental conditions as outlined by Furchgott (1967) and recognize changes in regression coefficients when calculating pA2 or KB values. Thus, observed relative potencies of agonists could equally depend upon drug disposition factors unless these variables are eliminated.

Since this is true for isolated tissues suspended in a fixed volume of physiological salt solution, distribution 28

and metabolism iii vivo will be expected to have a much greater influence on pharmacological responses (Ariens,

196 4). Therefore, the use of in vivo relative potencies

as a measure of receptor type is less valid than in vitro

studies of the same nature.

According to Furchgott (1955, 1967), after eliminating

the various factors which influence amine responses, the

activity of a given agonist in isolated tissues should

accurately reflect its true activity at the receptors as

long as responses are measured at equilibrium levels.

Implicit in this is the assumption that the concentration

of agonist in the region of the receptors is equal to or

some constant fraction of that added to the bath for all

tissues studied. In cases where the concentration of drug

in the hypothetical "biophase" is not equal to that in

the external medium, the true dissociation constant can

be found by multiplying the apparent dissociation constant

by the distribution coefficient of the drug between bath

fluid and receptor regions (Furchgott, 1955). Since, by

present techniques, it is not possible to determine such

distribution coefficients, the only measures of agonist

activity are apparent potency or apparent dissociation

constants (Furchgott, 1966; Furchgott and Bursztyn, 1967).

Distribution coefficients in different tissues are not

expected to be the same if the drug must pass through

diffusive tissue barriers to reach its site of action

(Stein, 1967) , even though responses to agonists are 29 relatively rapid in all tissues. Therefore, orders of potencies obtained from a series of agonists, even after exclusion of the several complicating factors, can only be apparent.

When antagonists are used to study receptors, the same arguments are valid. As recognized by Furchgott (1955,

1967) , calculated KB values are only apparent since the concentration of antagonist at the receptors is not known.

Even though appropriate time may be allowed for equilibrium to be established, the only parameter available to measure is response to an agonist and this gives no indication of the concentration of antagonist in the region of the recep tors. Therefore, the "selectivity" exhibited by some antagonists may only reflect their abilities to attain effective concentrations at receptor sites in some tissues and not in others. The factors which affect such penetra tion involve the physico-chemical properties of the drug in question and/or the nature of the tissue membranes which must be crossed by the antagonist to reach its site of action.

For these reasons, beta receptors of different tissues should not be considered heterogeneous until all factors affecting access of the drugs to these receptors are well understood or can be controlled. 30

Statement of the Problem

It has long been recognized that many biological drug sensitive sites are stereoselective (Cushny, 1926).

Cushny suggested that the use of optical isomers should yield valuable information regarding drug-receptor inter actions. In discussing the direct relationship between racemic potency and isomeric-activity-ratio, Pfeiffer

(1956) proposed that this ratio should give some indica tion of receptor conformation.

Stereochemical selectivity of alpha and beta adrener gic responses is well recognized (Ariens, 1967). When considering configuration around the 3-carbon atom of the catecholamine structure, the isomers with 1 R stereo chemistry are always more active than the corresponding antimers (Patil et al., 1970a).

After considering data regarding potencies of nore pinephrine. isomers from several sources, Patil (1969) suggested that, under the proper experimental conditions, tissues with identical receptor types should generate identical activity differences between optical isomers of agonists and antagonists with which these receptors interact. Subsequently, Patil et al. (1970b) observed that, from several tissues, even though the potencies of the individual isomers displayed 100 fold variations, the isomeric-activity-ratios of norepinephrine for activation of alpha receptors are identical. This indicated that 31 the alpha receptors of these tissues possess identical abilities to interact with the isomers of norepinephrine.

The obvious conclusion was that the alpha receptors in these tissues are homogeneous, a suggestion also made by

Furchgott (1967) on the basis of apparent KB values for phentolamine.

At the same time, Patil et a l . (1970b) reported that activity ratios between (-)- and (+)-norepinephrine for activation of beta receptors in several tissues displayed considerable variation. On this basis, it appeared that there were at least three different types of beta recep tors. Of the tissues studied, rat atria and bovine iris sphincter clearly belong in separate categories with isomeric-activity-ratios of 3000 and 7, respectively.

Surprisingly, a third group of tissues with intermediate and similar isomeric-activity-ratios included guinea-pig atria and trachea. Thus, although these two tissues reacted differently to many available "selective" agonists and antagonists and were said to possess different types of beta adrenergic receptor, results from optical isomers of norepinephrine indicated that their beta receptors might be similar. How could such divergent conclusions be explained?

Results from optical isomers are expected to be better than those from single agents. In other words, when racemic mixtures or only one of the resolved isomers 32 are studied, the objections previously raised about knowledge of "biophase" concentrations are fully valid.

The activities of these compounds in different tissues will depend upon their physico-chemical properties which influence distribution coefficients and diffusion to the active sites. Since optical isomers are expected to have identical physico-chemical properties, their use controls the factors which influence access (nonselective diffusion) to the receptors. At equilibrium, the concen tration of each isomer in the "biophase" will become equal to or some fraction of that applied to the external medium. Even though this relationship will vary between tissues for single agents, the proportional concentration of the optical isomers should remain constant. Therefore, knowledge of the "biophase” concentrations is not absolutely necessary. Hence, the isomeric-activity-ratio should be a relatively better criterion in differentiating adrener gic receptors.

However, although norepinephrine possess good beta receptor potency, it is not an ideal agent for studying these receptors. Its high affinity for alpha receptors, adrenergic neuronal membrane uptake sites, catechol-o- methyltransferase and monoamine oxidase could limit its usefulness. Even after attempts to eliminate these factors, there is no way to determine their effectiveness in each experiment. Furthermore, the activity of an 33 agonist is not only related to its affinity for the recep tor, but to its "intrinsic activity" or ability to induce a response once effective combination occurs (Ariens, 1964).

For these reasons, a more ideal agonist for studying beta receptors is isoproterenol. In view of these considera tions, the possibility that beta receptors of guinea-pig atria and trachea are similar is investigated utilizing optical isomers of the more specific agonist, isoproterenol.

Additional results are obtained from the optical isomers of some beta adrenergic~~receptor antagonists. Ideally, all drugs should have high potencies and small enough differences between their optical isomers to minimize non specific effects of their less potent (+) isomers.

The specific aim of this study is to critically evaluate the nature of beta adrenergic receptors of guinea-pig atria and trachea with the aid of activity differences between optical isomers of agonists rnd

antagonists. Additional experiments were desj to

investigate and define the role of some of the variables which may influence these activity differences. The speci

fic experimental goals may be outlined as follows:

.1) To determine isomeric-activity-differences for

the (-) and (+) isomers of isoproterenol, norepinephrine

and epinephrine.

2) To study some of the factors which could alter

the observed isomeric-activity-differences of the agonists. 34

By utilizing appropriate inhibitors, the influence of the

enzymes COMT and phosphodiesterase have been determined.

3) To determine pAx values for several beta adrener

gic antagonists and their isomeric-activity-differences in

the two tissues.

4) To study some of the factors which could influence

the observed isomeric-activity-differences of the anta

gonists. Experiments have been performed to evaluate the

rates of onset of blockade in the two tissues and to deter

mine if (-) and (+) isomers of the same blocker compete

for the same sites.

Under proper experimental conditions, if beta

adrenergic receptors in guinea-pig atria and trachea are

similar, activity differences between the optical isomers

of beta receptor agonists and antagonists are expected to

be similar. CHAPTER II

METHODS AND MATERIALS

General considerations

Albino guinea pigs (Beau Manor Farms, Cleveland, 0.),

weighing 300 to 700 g, were killed by a sharp blow on the

head. Right atria or trachea were removed, cleaned free

of excess tissue and suspended in water-jacketed (37-38°C)

10-ml tissue baths containing a physiological salt solution

of the following composition: NaCl, 118 mM; KC1, 4.7 mM;

CaCl2 *2H20, 1.9 mM; NaHC03 , 25 mM; MgCl2 *6H20, 0.5 mM;

Na^PO^-I^O, 1 mM and glucose, 11 mM. The chemicals were

dissolved in double distilled demineralized water. Two

such tissues were mounted and tested simultaneously. The

baths and stock salt solution were aerated with a mixture

of oxygen (95 percent) and carbon dioxide (5 percent).

Responses were recorded on a Grass model 7 polygraph via

force-displacement transducers (FT-0 3).

Cumulative dose-response effects of the agonists were

obtained by increasing the concentrations by a factor of

about 3 while the previous dose remained in contact with

the tissue (Van Rossum, 1963). This was done only after

' the effects of that does had reached maximum and remained

constant. Unless stated otherwise, 15 to 30 min were

35 36 required for completion of all dose-response curves. All

other agents were added to the bath in a volume of 0.1 ml

and allowed to interact with the tissue for fixed periods

of time.

Isolated right atria

Spontaneous atrial contractions were recorded

together with atrial rate which was monitored with Grass

model 7P4A tachographs to determine when maximum responses

occurred to a given concentration of agonist. The amount

of tension exerted on each atrium was the minimum needed

to obtain a pen deflection of about 0.5 cm per beat at the

highest preamplifier sensitivity without recording back

ground noise. Each tissue was allowed to equilibrate for

one hr prior to addition of any drug and washings were

made at 15 min intervals during this period. At this

time, atrial rates usually did not vary by more than 5 to

10 beats per minute. Agonist-induced increases in rate

from baseline ranged from 100 to 200 beats per minute.

I sol at ed •tracheal' strips

Trachea were cut in a spiral fashion, each turn sepa

rated by 3 to 4 cartilage segments (Patterson, 1958;

Constantine, 1965). The strip was approximately halved

and each half mounted in a tissue bath. Strips were

allowed to equilibrate for two hr prior to the addition of

any drug and washings were made at 15 min intervals during

this period. Resting tension was maintained at 5 g 37 during equilibration and subsequent drug incubations.

Relaxation of the tracheal strip was studied after par- — 7 — 6 tial contraction with carbachol, 3 X 10 or 10 M

(approx 30 percent of its own maximum). As previously

determined, this contraction reaches a maximum (1 to 3 g

developed tension) in 10 to 15 min and remains constant

for at least one hr. In order to keep all drug contact

periods constant, cumulative dose-response curves to

beta receptor agonists were begun 15 min after addition

of carbachol to the bath.

Use of optical isomers of agonists

Unless stated otherwise, a cross-over design was

utilized in which each isomer was tested first on about

one-half of the tissues in each series of experiments.

A cumulative dose-response curve for the other isomer of

the same agonist was obtained after washing the tissue

for 45 to 50 min after the first dose-response curve and

allowing it to equilibrate 15 min after the last wash

before any drug was added. Washing consisted of complete

replacement of the bath fluid with fresh physiological

salt solution 10 to 11 times during this period. Usually,

both isomers of the same agonist were tested simultaneous

ly on separate tissues.

Since various factors are known to influence the

pharmacological activities of the isomers (Furchgott,

19 67), it is desirable to determine isomeric-activity-

differences in the presence of substances which can reduce these effects (Patil, 1969). Hence, dose-response curves

to the isomers of norepinephrine and epinephrine in each

tissue were obtained in the presence of the following drugs

1) tropolone, 3 X 10"^ M, to inhibit the enzyme catechol-o-

methyltransferase, 2) phentolamine, 10“^ M, to block alpha

receptors and 3) cocaine, 1 0 “5 m, to prevent adrenergic

neuronal membrane uptake. The total tissue contact times

for these agents were 45, 35 and 25 min, respectively.

Dose-response curves for isomers of isoproterenol were

made in the presence of the same concentrations of tropo

lone and phentolamine, which were allowed to interact

with the tissues for 35 and 25 min, respectively. When

cocaine was utilized, it did not alter isomeric-activity-

differences of isoproterenol in either tissue. When it

was desired to determine the effects of tropolone, this

agent was substituted with an equal volume of 0.9 percent

saline and the appropriate incubation times remained the

same.

Phentolamine lowered the atrial rate by 50 to 100

beats per min and did not alter initial tracheal tone.

Although the effects of alpha receptor activation on

chronotropic responses in guinea-pig atria are nil

(Krell and Patil, 1969) , phentolamine was added to this

. tissue in order to control possible drug-drug interactions.

Its rate depressant action does not influence responses

to the catecholamines and was considered advantageous in 39

that it produced a more stable baseline at lower rates,

thereby allowing a larger range over which to study rate

increasing effects of the agonists.

Trendelenburg (1968) has shown that the degree of

potentiation of responses to catecholamines caused by

cocaine can be underestimated when the tissues contain

intact stores of norepinephrine. For this reason, where

indicated, experiments were performed on tissues taken

from guinea pigs which had been pretreated with reserpine

(5 mg/kg i.p.) 16 to 2 4 hr previously.

The effects of theophylline on atrial responses to

isoproterenol stereoisomers were determined by construct

ing cumulative dose-response curves to the agonists in

the presence of the following drugs: 1) theophylline,

10"3 m , 2) tropolone, 3 X 10”^ M and 3) phentolamine,

1 0 "5 m. In some experiments, incubation periods were 45,

35, and 25 min, respectively. In others, they were 25,

45, and 35 min, respectively. This change in order of

drug addition did not alter the results and values from

both samples were pooled. Theophylline alone increased

atrial rates by 75 to 100 beats per minute. Since phen

tolamine reduced rates by about the same extent, there was

no problem in maintaining an adequate range over which to

, study positive chronotropic actions of isoproterenol.

Responses to the agonists were usually calculated

as a percent of the maximum obtained in each dose-response 40

curve. Isomeric-activity-differences were determined at

ED50 levels for each tissue and expressed as differences

between the negative log molar ED50 values. The antilog

of this value is the isomeric-activity-ratio.

Use of optical isomers of antagonists

TWo cumulative dose-response curves to (-)-isopro

terenol were constructed on each tissue, one before and

one in the presence of a beta receptor antagonist.

Between curves, each tissue was washed as described for

the use of agonists. Three to four concentrations of

antagonist were used in evaluating blocking effects, one

concentration and antagonist on each tissue. Usually,

both isomers of the same antagonist were tested simul

taneously on separate tissues.

Antagonists were allowed to interact with the tissues

for 45 min prior to construction of the second dose-response

curve. In trachea, this was 30 min before adding carbachol.

An equal volume of 0.9 percent saline was added 45 min

before control responses were obtained.

The alpha receptor blocking action of beta receptor

antagonists is slight and not enough to block the entire

alpha receptor population (Patil et. al., 1968) . Since

phentolamine does not interfere with establishment of

- keta adrenergic blockade (Krell and Patil, 1969), a con

centration of 10“5 m of this agent was added to the bath

55 min prior to obtaining both dose-response curves to 41

the agonist.

Essentially the same experimental design was followed

in studying the rate of onset of beta adrenergic blockade.

Selected concentrations of (-)- and (+)-alprenolol were

allowed to interact with each tissue for 3, 10, 25, 45, 65

or 120 minutes. For control purposes, complete cumulative

dose-response curves were first obtained at the same time

intervals after adding an equal volume of 0.9 percent

saline. In trachea, phentolamine (10“5 M) was added 10 min

prior to the start of incubation periods. This agent was

not utilized in atria. Dose-response effects of the agonist

in the presence of antagonist were obtained with two concen

trations of agonist which were predicted to produce re

sponses below and above the ED50 obtained in the control

curve on each tissue. This procedure was necessary since

responses to isoproterenol in both tissues develop slowly

and it is desirable to determine the degree of blockade as

closely as possible to the designated time of antagonist

incubation. Even with this technique, 5 to 10 min were

required for equilibrium responses to be established.

Since the antagonists used in this study have been

assumed to be competitive, their effects were analyzed

according to the method of Arunlakshana and Schild (1959).

. Simple competitive antagonism follows the equation:

lOg (x - 1) = log 1/Kb - npAx where x = dose ratio, = dissociation constant of the

antagonist-receptor complex, pAx = negative log of the

antagonist concentration and n is a constant equal to the

slope of the regression line of a plot of log (dose ratio

- 1) vs. pAx. The theoretical value of n is -1 for com

petitive antagonism. When slope values are less than -1,

the Kb values increase with increasing concentrations of

antagonists and dissociation constants cannot be deter

mined.

All points obtained by plotting log (dose ratio - 1)

vs. molar concentration of antagonist were connected

visually. In this case, concentrations were plotted on a

log scale and resulting slopes have a theoretical value

of +1. Activity differences between isomers of the

antagonists were determined at pA]_g levels and expressed

as the isomeric-activity-difference [(pA^g °f (“) isomer)

- (pA^g of (+) isomer)] . Isomeric-activity-ratios refer

to the antilog of this value. Responses to isoproterenol

were calculated as a percent of the maximum obtained in the

control curve and negative log molar ED50 values were used

for calculating dose ratios antilog [(negative log molar

ED50 without antagonist) - (negative log molar ED50 with

antagonist)] . For studies on rate of onset of blockade,

* only the absolute differences between negative log molar

ED50 values were calculated and expressed as log (dose

ratio). 43

Since changes in sensitivity after the first curves

are different for the two tissues (Table 3), where

necessary, the dose ratios were corrected for desensitiza

tion. This correction was not done routinely because

isomeric-activity-differences are not altered by this

procedure.

Standard errors of the mean (S.E.M.) were calculated

for all samples and 9 5 percent confidence intervals (C.I.)

for isomeric-activity-differences of the agonists. Slopes

of the regression lines in pAx determinations were com

puted by linear regression analysis (Freund et al., 1960).

Differences between two means were found using Student's

t test and a p value of ^.05 was regarded as being sig

nificant.

Drugs and Solutions

The drugs used in this study were made fresh on the

day of each experiment and kept refrigerated until 5 min

before use. The signs (-) and (+) refer to the direction

of rotation of polarized light, levo and dextro, respec

tively. The sign (±) refers to the racemic mixture.

Dilutions of (-) isomers of the agonists were made from

10”2 m refrigerated stock solutions prepared in 0.9 per

cent saline with 0.05 percent sodium metabisulfite to

. minimize spontaneous oxidation. A small amount of

dilute HC1 was required for the dissolution of (-)-epin

ephrine and (-)-norepinephrine. The (+) isomers of the

agonists and desoxy-isoproterenol were prepared daily 44

TABLE 3

Changes in Sensitivity of Guinea-pig Tissues Obtained

from Separate Control Experiments

Negative log molar ED50 of Desensitization^ Tissue (-)-isoproterenol3, with S.E.M. N c in log units 1st curve 2nd curve with S.E.M.

Atria*3 8.05 ± 0.1 7.66 ± 0.12 7 0.39 ± 0.04

Trachea 8.09 ± 0.09 7.95 ± 0.09 8 0.14 ± 0.03

aObtained at 45 min incubation periods in the presence of phentolamine and absence of beta adrenergic antagonist.

^Average atrial rate before 1st and 2nd curves were 156 (S.E.M. ± 6) and 173 (S.E.M. ± 9) beats/min, respectively.

CN = number of observations.

^Log Difference = (negative log molar ED50 of 1st curve) - (negative log molar ED50 of 2nd curve). 45 in the same medium except HCl was not needed. Dilutions of carbachol were made from 10"2 M refrigerated stock solutions prepared in 0.9 percent saline. Other drugs were prepared in 0.9 percent saline, with a small amount of dilute HCl required to dissolve (±)-practolol. All strengths are expressed as molar concentrations, aminophylline in terms of theophylline content.

The following drugs were used: (-)-1-(o-allylphenoxy)

-3-isopropylamino-2-propanol tartrate monohydrate

(alprenolol, H56/28); (+)-1-(o-allylphenoxy)-3-isopropylamino-2-propanol HCl (alprenolol, H56/28); (-)- and (+)

- 1 - (4-nitrophenyl)-2-isopropylaminoethanol HCl (INPEA);

(-)- and (+)-4-(2-isopropylamino-l-hydroxyethyl) methane- sulfonanilide HCl (sotalol, MJ 1999); (±)-4-(2-hydroxy-

3-isopropylaminopropoxy) acetanilid (practolol, AY 21011);

(-)-isoproterenol-(+)-bitartrate dihydrate; (+)-isopro terenol- (+) -bitartrate; 3-desoxy-isoproterenol HCl; (-)- norepinephrine (base); (+)-norepinephrine-(+)-bitartrate;

(-)-epinephrine (base); (+) -epinephrine- (-) -bitartrate; tropolone; phentolamine methanesulfonate; cocaine HCl; reserpine (Serpasil, Ciba); carbachol chloride and aminophylline. The same samples of the isomers of agonists and antagonists were used for the entire study. Sources and formula weights of the chemicals are listed in Table 12 of the appendix. CHAPTER III

RESULTS

Isomeric-activity-differences of beta receptor agonists

Cumulative dose-response curves to (-) and (+) isomers of isoproterenol, norepinephrine and epinephrine in atria

and trachea taken from reserpine-pretreated and normal

animals are represented in Figures 1, 2 and 3. Isomeric-

activity-dif ferences are shown between the horizontal

arrows connecting the curves for each pair. Table 4

summarizes the data taken from these curves. The maximum difference in ratio between the two tissues is about 2.5

fold (0.4 log units). Reserpine-pretreatment had no

influence on the activity difference between the isomers,

although it did result in a parallel shift to the left of the dose-response curves to all agonists in trachea and to epinephrine in atria. The effects of norepinephrine

isomers were not tested in atria taken from normal animals.

Potentiation of responses of tracheal strips to isopro

terenol by short-term pretreatment with reserpine indicates

that a nonspecific supersensitivity, as that described for

rabbit aorta (Hudgins and Fleming, 1965), is also operative

in this tissue.

46 FIGURE 1

Log-dose-response curves for (-)- and (+)-iso proterenol obtained from atria and trachea taken from normal and reserpine-pretreated guinea pigs. Numbers between the horizontal arrows connecting the curves are

isomeric-activity-differences [(negative log molar ED50

of (-) isomer - (negative log molar ED50 of (+) isomer)] .

Values enclosed in parentheses are 95 percent C.I. and vertical lines indicate S.E.M. All curves were obtained

in the presence of tropolone and phentolamine. Each

curve for normal tissues represents 8 observations and each curve obtained on tissues from reserpine-pretreated

animals represents 4 observations.

47 % % POSITIVE CHRONOTROPIC RESPONSE % RELAXATION 100 100 40 60 40 80 20 80 60 20 • • — • — (-)- (-)- SPOEEO * RESERPINE * ISOPROTERENOL ISOPROTERENOL PRETREATEO >9

( 253-2 •8 — 2

92(2 301(2

2 85

73(2 ) AGONIST (M)AGONIST UNAPG RCEL STRIP TRACHEAL GUINEA-PIG

80-3

72-3

68-2

UNAPG ATRIA GUINEA-PIG 4 0

78 •7 ) )

) (♦) - SPOEEO - RESERPINE - ISOPROTERENOL - ) ♦ ( o o —o ISOPROTERENOL - ) ♦ ( o o— 65 •6 PREThEATED '4 43 FIGURE 2

Log-dose-response curves for (-)- and (+)-nore pinephrine obtained from trachea taken from normal and reserpine-pretreated guinea pigs. Atria were taken from reserpine-pretreated animals only. Numbers between the horizontal arrows connecting the curves are isomeric- activity-dif ferences {(negative log molar ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer)] .

Values enclosed in parentheses are 95 percent C.I. and vertical lines indicate S.E.M. All curves were obtained in the presence of tropolone, phentolamine and cocaine.

Each curve from atria and trachea taken from reserpine- pretreated animals represents 8 observations and curves from trachea taken from normal animals represent 6 observations.

49 % % POSITIVE CHRONOTROPIC RESPONSE % RELAXATION 100 100 40 40 80 80 60 60 20 1-8 o • ------OEIEHIE —O (+ NOREPINEPHRINE - ) + ( O RESERPINE - NOREPINEPHRINE O— - 1 ) + O O RESERPINE - NOREPINEPHRINE - ) - NOREPINEPHRINE ( - ) • - ( o d e t a e r t e r p 182- ) 0 4 -2 2 8 (1 5 1 2 214- 46) 6 4 -2 4 1 (2 0 3 2

NEA-PI A - RESERPI D E T A E R T E R P E IN P R E S E R - IA R T A IG P - A E IN U G UNAPGTAHA STRIP TRACHEAL GUINEA-PIG AGONIST (M) AGONIST ------*s \* d e t a e r t e r p FIGURE 3

Log-dose-response curves for (-)- and (+)-epine phrine obtained from atria and trachea taken from normal and reserpine-pretreated guinea pigs. Numbers between the horizontal arrows connecting the curves are isomeric-activity-dif ferences [(negative log molar ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer)] .

Values enclosed in parentheses are 95 percent C.I. and vertical lines indicate S.E.M. All curves were obtained in the presence of tropolone, phentolamine and cocaine.

Each curve represents 8 observations.

51 % POSITIVE CHRONOTROPIC RESPONSE % RELAXATION 100 100 80 40 40 60 80 60 20 « • ------IA R T A G I P - A E N I U G )-EPI NE IN E R H IN P P E R E IN S P E E R - - ) + E ( IN R H P o E IN — P E — o - ) + ( O o E IN P R E S E R - E IN R H E P N I E R N I H P P E E N - I P E ) - - ( ) - • ( • D E T A E R T E R P .515-.4 — 1.75(156-1.94) 162- ) 8 9 -1 2 6 (1 0 8 1 IP R T S L A E H C A R T G I P - A E N I U G ------AGONIST (M) AGONIST t *- - D E T A E R T E R P 52 Effects of Optical Isomors of Beta Receptor Agonists on Guinea-pig Atria and Tracheal Strips

Isolated guinea-pig Isolated guinea-pig right atriaa ______tracheal strip* Differ Agonist Negative log Isomeric-activity “ Negative log Isomeric-activity ence molar ED50 difference*3 N" molar EDSO difference" N^ between ______with S.E.M. (95% C.I.)______with S.E.M. (95X C.I.)______tissues0 Reserpine-pretreated tissue: (-)-isoproterenol 8.67 t 0.04 8.90 ± 0.06 3.01 4 2.69 4 0.32 (+)-isoproterenol 5.66 t 0.08 (2.72-3.30) 6.21 ± 0.03 (2.53-2.85)

(-)-norepinephrine 7.47 t 0.05 7.03 t 0.04 2.70 8 2.30 8 0.40 (+)-norepinephrine 4.77 ± 0.07 (2.53-2.87) 4.73 t 0.04 (214-2.46

(-)-epinephrine 7.35 ± 0.06 7.98 t 0.07 1.80 8 1.46 8 0.34 (+)-epinephrine 5.55 t 0.04 (1.62-1.98) 6.53 t 0.06 (1.41-1.50)

Normal tissue: (-)-isoproterenol 8.65 i 0.05 8.50 ± 0.05 2.92 8 2.73 8 0.19 (+)-isoproterenol 5,73 ± 0.04 (2,80-3.04) 5.77 ± 0.05 (2.68-2.78)

(-)-norepinephrine Mesa _____ 6.67 t 0.09 - 2.15 6 (•*•) -norepinephrine 4.52 ± 0.04 (1 .82-2 .48)

(-)-epinephrine 7.11 ± 0.05 7.83 ± 0.03 1.75 8 1.47 8 0.28 (+)-epinephrine 5.36 i 0.04 (1.56-1.94) 6.36 + 0.04 (1.40-1.54)

^Tissues were exposed to tropolone/ phentolamine and cocaine. In the case of isomers of isoproterenol, cocaine was not used. See Methods for further details. ^Isomeric-activity-difference ■ [(Negative molar log ED50 of (-)-isomer) - (negative log molar ED 50 of (+)-isomer)] . cDifference between tissues - (isomeric-activity-difference in atria) - (isomeric-activity- dif ference in trachea). °N — number of observations. 54

Usually, the maximum response obtained with (+)- isoproterenol and (+)-epinephrine was maximal for the respective isomer in both tissues since subsequent addition of the (-) isomer in a concentration about 3 times that necessary for its own maximum did not result in further increase in response. Nor was there any difference in maximum response obtained with (-) and

(+) forms on a single tissue. For atria, addition of

(-)-norepinephrine at the height of the maximum response to (+)-norepinephrine resulted in a further increase in rate. The maximum obtained with the (+) isomer was 96 percent (S.E.M. ±0.7) of that produced by further addition of its antimer. In trachea from normal and reserpine-pretreated animals, the maximum obtained with

(+)-norepinephrine was 99 percent (S.E.M. ±0.4) of its

(-) isomer. Responses to (+)-norepinephrine were calculated using the maximum effects induced by the sub sequent addition of (-)-norepinephrine.

Since the experimental design was such that both isomers of a single agonist were tested on the same tissue, any desensitization in the second curve may obscure the true activity difference. Usually, the isomeric- activity-dif ference was larger when the (-) isomer was tested first. It is possible that relatively more (+) isomer remains in the tissue at the beginning of the 55 dose-response curve to its antimer, thereby producing specific antagonism and/or desensitization. As a result, the isomeric-activity-difference will appear smaller.

Although atrial rates before the second dose-response curves were usually greater than those before the first curves, average rates before all dose-response curves for the (-) isomers were not different from those before all curves for the (+) isomers. When only first curves obtained in each sample are considered, isomeric-activity- differences are not drastically changed (Table 5). Dif ferences between ED50 values obtained from the first curves and those of the average of all curves for the same isomer were never greater than 0.2 log units.

Since orders of potencies of beta receptor agonists are considered by others as valuable tools in differen tiating receptors, dose-response curves for the (-) forms of agonists used in this study are compared in Figure 4.

The two distinctly different orders are consistent with previously mentioned observations by other investigators.

Influence of tropolone on

isomeric-activity-differences of agonists

To determine the influence of COMT on responses to the catecholamine isomers, the inhibitor, tropolone, was excluded from some experiments. The results are illus trated in Figures 5 through 8 and summarized in Table 6.

Only responses to the isomers of isoproterenol are TABLE 5 Effects of Optical Isomers of Beta Receptor Agonists Obtained from the First

Curves of Each Experiment in Guinea-pig Atria and Trachea.

Isolated guinea-pig Isolated guinea-pig right atriaa tracheal strip® Differ Agonist Negative log Isomeric-activity Negative log Isomeric-activity ence molar ED50 difference" Nd molar ED50 difference" Nd between ± S.E.M. i S.E.M. tissues0 Reserpine-pretreated tissue: (-)-isoproterenol 8.69 1 0.05 2 8.91 + 0.1 2 2.89 2.70 0.19 (+)-isoproterenol 5.80 1 0.05 2 6.21 ± 0.08 2

(-)-norepinephrine 7.55 i 0.05 4 7.03 ± 0.06 4 2.79 2.32 0.47 (+)-norepinephrine 4.76 1 0.03 4 4.71 t 0.04 4

(-)-epinephrine 7.47 t 0.07 4 8.05 i 0.09 4 1.86 1.60 0.26 (+)-epinephrine 5.61 i 0.05 4 6.45 i 0.08 4

Normal tissue: (-)-isoproterenol 8.75 ± 0.04 4 8.48 1 0.09 4 2.98 2.71 0.27 (+)-isoproterenol 5.77 t 0.06 4 5.77 t 0.08 4

(-)-norepinephrine -- - 6.63 i 0.1 3 2 09 (+)-norepinephrine -- - 4.54 1 0.07 3

(-)-epinephrine 7.25 ± 0.02 4 7.86 ± 0.04 4 1.81 1.46 0.35 (+)-epinephrine 5.44 t 0.02 4 6.40 i 0.08 4

^Tissues were exposed to tropolone, phentolamine and cocaine. For the isomers of isoproterenol, cocaine was not used. ^Isomeric-activity-difference “ [(negative log molar ED50 of (-)isomer)-(negative log molar ED50 of (+) isomer)] . cDifference between tissues - (isomeric-activity-difference in atria)-(isomeric-activity-difference in trachea.) "N » number of observations. FIGURE 4

Log-dose-response curves for (-) forms of iso proterenol, norepinephrine and epinephrine obtained from atria and trachea taken from reserpine-pretreated guinea pigs. All curves were obtained in the presence of drug incubations listed in Methods. Numbers in parentheses are negative log molar ED50 values with

S.E.M.; n = number of observations. Vertical lines indicate S.E.M.

57 % % POSITIVE CHRONOTROPIC RESPONSE % RELAXATION 100 100 40 60 80 40 80 60 20 O H )I - >(-1NE - ( I> P )E - ( > 0 IS ) 1 - - POTENCIES OF ORDER - RE O POTENCIES OF ORDER )IO>( ) X-1EPI P E 1 - X E -)N —)( ( > ISO UNAPG RCEL TI - EEPN PRETREATED RESERPINE - STRIP TRACHEAL GUINEA-PIG

A - RESERPI D E T A E R T E R P E IN P R E S E R - IA R T A G I P - A E N I U G AGONIST (M) NEPHRI ) I P E ( E IN R ) H P E E N IN ) ( O P E IS E - IN ( R ) H P E L - O IN P N ( E E R R O E N T O - R * ) P • O - IS ( - ) ■a - ( ■« FIGURE 5

Log-dose-response curves for (-) and (+)-isopro terenol obtained with and without tropolone from trachea taken from normal guinea pigs. Numbers between the horizontal arrows connecting the curves are isomeric-

activity-dif ferences [(negative log molar ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer)] .

Values enclosed in parentheses are 95 percent C.I. and vertical lines indicate S.E.M. Each curve was obtained in the presence of phentolamine and represents 8 obser vations .

59 % % RELAXATION 100 0 4 20 0 8 0 6 » • ------IORTRNL IH RPLN (3 TROPOLONE ( WITH ISOPROTERENOL - ) - ( # (-) SPOEEO WTOT RPLN C------(♦) IORTRNL IHU TROPOLONE WITHOUT -ISOPROTERENOL ) ♦ ( C------0 TROPOLONE WITHOUT ISOPROTERENOL - ) - ( C 10-* UNAPG RCEL STRIP TRACHEAL GUINEA-PIG i 73(2. 78)- ) 8 .7 2 - 3 .6 2 ( 3 .7 2 I "M) O ) 0"5M AGONIST (M) 30(2. 2. ) 1 .4 -2 9 .1 2 ( 0 .3 2 ------IORTRNL IHTOOOE O"5M) " lO x 3 ( TROPOLONE WITH -ISOPROTERENOL ) + ( 0 io- io- or o FIGURE 6

Log-dose-response curves for (-)- and (+)-isopro terenol obtained with and without tropolone from atria taken from normal guinea pigs. Numbers between the horizontal arrows connecting the curves are isomeric- activity-dif ferences [(negative log molar ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer)]

Values enclosed in parentheses are 95 percent C.I. and vertical lines indicate S.E.M. All curves were made in the presence of phentolamine. Curves obtained in the absence of tropolone represent 7 observations and those with tropolone represent 8 observations.

61 % % POSITIVE CHRONOTROPIC RESPONSE 100 0 4 0 6 eo 20 • • SPOEEO WTOT RPLN o- o TROPOLONE WITHOUT ISOPROTERENOL - ) - ( • • ----- 8

IORTRNL IH RPLN (3ilO't) o tW) ' O l i 3 ( TROPOLONE WITH -ISOPROTERENOL ) - I I0*» 10" • ------2.92 AGONIST AGONIST (M) UNAPG ATRIA GUINEA-PIG ( 2 . 0 6 53(2. 2. ) 5 .7 -2 1 .3 2 ( 3 .5 2 - 3 . 4 0 ) ------SPOEEO WTOT TROPOLONE WITHOUT ISOPROTERENOL - ) ♦ ( o - SPOEEO WT TOOOE 3 ( TROPOLONE WITH ISOPROTERENOL - ) ♦ ( 0 10-' i 0*5M) * I0 or FIGURE 7

Log-dose-response curves for (-)- and (+)-nore pinephrine obtained with and without tropolone from atria taken from reserpine-pretreated animals. Numbers between the horizontal arrows connecting the curves are isomeric-activity-dif ferences [(negative log molar

ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer [] . Values enclosed in parentheses are 9 5 percent C.I. and vertical lines indicate S.E.M. All curves were made in the presence of phentolamine and cocaine. Curves obtained in the absence of tropolone represent 4 observations and those with tropolone represent 8 observations.

63 % POSITIVE CHRONOTROPIC RESPONSE 100 0 4 0 8 0 6 20 © © ------,-io NEPHRI THOUT TROPOLONE N O L O P O R T T U O H IT W E IN R H P E IN P E R O N - ) - ( © NEPHRI TH TROPOLONE (3xlO"5M) M 5 " O l x 3 ( E N O L O P O R T H IT W E IN R H P E IN P E R O N - ) - { 0 IE-I TI - EEPN PRETREATED RESERPINE - ATRIA UINEA-PIG G i-s 2 7012 5J- 2 871 2 60 12 28 - 2 92)---- re GNS (M) AGONIST O )-NOREPI NE IN R H P E IN P E R O N - ) + ( O — O ------)-NOREPI NE IN R H P E IN P E R O N - ) + ( O 1-6 THOUT TROPOLONE N O L O P O R T T U O H IT W ) M * ‘ O l x 3 ( E N O L O P O R T H T I W i-s FIGURE 8

Log-dose-response curves for (-) and (+)-epine phrine obtained with and without tropolone from atria

taken from normal animals. Numbers between the hori

zontal arrows connecting the curves are isomeric-

activity-dif ferences [(negative log molar ED50 of (-)

isomer) - (negative log molar ED50 of (+) isomerf]

Values enclosed in parentheses are 95 percent C.I.

and vertical lines indicate S.E.M. Each curve was obtained in the presence of phentolamine and cocaine

and represents 8 observations.

65 \ \

GUINEA-PlO ATRIA

(-)- epinephrine w it h t r o p o l o n e ( 3 i i o " s ) (+J-EPINEPHRINE WITH TROPOLONE (3 x K T 9) (-)- epinephrine w it h o u t t r o p o l o n e 0 o (+1-EPINEPHRINE WITHOUT TROPOLONE 100 lli

o O 60 1.750.56-1.94 1.620.34-1.90)-

I 40

LjJ 20

1-7 10-' 10'9 »- i-s AGONIST (M)

or or TABLE 6

Effects of the Isomers of Catecholamines in the Absence and Presence of Tropolone

In the absence In the presence of tropolone of tropolone, 3 x 10"^ M Negative Isomeric- Negative Isomenc- Agonist log molar activity- log molar activity- ED50 differencec ED50 differencec + S.E.M. (95% C.I.) N l ± S.E.M. (95% C.I.) N<

Guinea-pig atriaa:

(-)-isoproterenol 8.01 + 0.09 2.53 7 8.65 ± 0.05e 2.92 8 (+)-isoproterenol 5. 47 + 0. 05 (2.31-2.75) 5.73 + 0.0 4e (2. 80-3.04)

(-) -norepinephrine*3 7.33 + 0.05 2.60 4 7.47 + 0.05 2.70 8 (+)-norepinephrine 4.73 + 0.06 (2.28-2.92) 4. 77 + 0.07 (2.53-2.87)

(-)-epinephrine 6.97 + 0.08 1.62 8 7.11 + 0.05 1.75 8 (+)-epinephrine 5.35 + 0.06 (1.34-1.90) 5.36 + 0.04 (1.56-1.94)

Guinea-pig trachea:

(-)-isoproterenol 7. 89 + 0.1 2. 30 8 8.50 + 0. 05e 2.73 8 (+)-isoproterenol 5.59 + 0.07 (2.19-2.41) 5.77 + 0.05 (2.68-2.78)

aTissue was exposed to phentolamine and cocaine when testing isomers of norepine phrine and epinephrine. Cocaine was not used with isoproterenol isomers. ^Values for (-)-and (+)-norepinephrine obtained in atria taken from reserpine- pretreated (5 mg/kg i.p., 16-24 hr prior) animals. cIsomeric-activity-difference = [(negative log molar ED50 of (-) isomer)- (negative log molar ED50 of (+) isomer)] . dN = number of observations. eDenotes significant potentiation by tropolone. 68 significantly potentiated by the addition of the inhibitor/ apparently in a stereoselective manner. In the presence, of tropolone, dose-response curves to (-)-isoproterenol in both tissues are shifted to the left by about 0.6 log units. The curves to (+)-isoproterenol were altered in a similar manner by 0.2 to 0.3 log units. As a result, isomeric-activity-differences for isoproterenol for in creased by about 0.4 log units in the two tissues.

Influence of theophylline on the isomeric-activity-

difference of isoproterenol in guinea-pig atria

Figure 9 illustrates how responses to isoproterenol in guinea-pig atria are influenced by tropolone and theophylline. Depending upon the experimental condi tions, there is a progressive shift of the curves for both isomers of isoproterenol from right to left. For the (-) form, the negative log molar ED50 values increase from

8.01 through 8.65 to 9.20, a maximum potentiation of 1.2 log units. Similar values for the (+) isomer are 5.47,

5.73 and 6.27, representing a maximum potentiation of 0.8 log units. Without tropolone and theophylline, the isomeric-activity-difference is 2.5 log units. This value is increased to 2.9 log units by tropolone and remains un altered by further addition of theophylline. If dose- response curves to the isomers obtained in the presence of only tropolone were not included in the comparison, the

effects of theophylline would appear stereoselective. FIGURE 9

Log-dose-response curves for (-)- and (+)-isopro

terenol obtained with and without tropolone, 3 X 10“^ X,

and/or theophylline, 10“^ M, from atria taken from normal

guinea pigs. Numbers between the horizontal arrows

connecting the curves are isomeric-activity-differences

[(negative log molar ED50 of (-) isomer) - (negative log molar ED50 of (+) isomer)] . Values enclosed in paren

theses are 95 percent C.I.; n = number of observations

for each pair. All curves were obtained in the presence

of phentolamine.

69 Guinea-pig Atria

% Positive Chronotropic Response 100- -O

60 2.53(2.31-2.75) P — 2.92 (2.80 -3.0 4 ) 2.93(2.86-3.00) 40

• - ( - ) - isoproterenol O—(+).- isoproterenol 20 n = 7

ml -10 -9 Agonist (M) 71

Isomeric-activity-differences of

beta receptor antagonists

Plots of log (dose ratio - 1) vs. molar concentra tion of the optical isomers of beta receptor antagonists are depicted in Figures 10, 11 and 12. Differences be tween each pair of isomers measured at pA^g levels are indicated by the number between the arrows connecting their regression lines. Tables 7 and 8 summarize the data used to construct these graphs. In all cases, the

(+) isomer is approximately l/100th as active as its (-) form. When isomeric-activity-differences from the two tissues are subtracted, the values (in log units) for alprenolol, sotalol and INPEA are 0.39, 0.16 and 0.31, respectively. Thus, there is a maximum of about a 2.5 fold difference between the ratios obtained on atria and trachea.

The isomeric-activity-differences obtained for INPEA and sotalol in tracheal strips are consistent with those reported for tracheal chains by Patil (1968). Slopes of the regression lines are also similar. However, sotalol is 10 times and INPEA 3 to 4 times more potent in the present studies. Here, incubation periods for the an tagonists are 15 min longer than in the previous experi ments .

When pA 2 or pA]_o values of only the (-) forms are considered, the order of blocking activities for the antagonists in atria is alprenolol>INPEA^sotalol, while FIGURE 10

Plots of log (dose ratio - 1) vs_. molar concentra tion of (-)- and (+)-alprenolol obtained from guinea-pig atria and trachea. (-)-Isoproterenol was used as agonist.

Numbers between the horizontal arrows connecting the regression lines are isomeric-activity-differences [(pA^o of (-) isomer) - (pAj_Q of (+) isomer)] . Slopes of the regression lines for (-) and (+) isomers from atria are

0.91 and 0.89, respectively, and from trachea are 0.64 and

0.72, respectively. Vertical lines indicate S.E.M. and values in parentheses are the numbers of observations.

The tissue incubation time for both antagonists was 45 min.

Asterisk denotes an asymmetric carbon atom in the struc tural formula. See Methods for further details.

72 LOG (DOSE RATlO-l) LOG (DOSE RATIO-1) O — _ ro ho w* O i* o u> O i ' i i r »'■» i i i i i mi i~i i t i i i r i i n[ i t t r -)

2 S m® m9 UNAPG RCEL STRIP TRACHEAL GUINEA-PIG

> Z g o LO2 -I s

CO FIGURE 11

Plots of log (dose ratio - 1) vs. molar concentra tion of (-)- and (+)-sotalol obtained from guinea-pig atria and trachea. (-)-Isoproterenol was used as agonist.

Numbers between the horizontal arrows connecting the re gression lines are isomeric-activity-differences ^(pA^o of (-) isomer) - (pA^g of (+) isomer)] . Slopes of the regression lines for (-) and (+) isomers from atria are 0.9 9 and 1.00, respectively, and from trachea are

0.65 and 0.57, respectively. Vertical lines indicate

S.E.M. and values in parentheses are the numbers of observations. Asterisk denotes an asymmetric carbon atom in the structural formula. The tissue incubation time for both antagonists was 45 min.

74 LOG (DOSE RATIO-1) LOG (DOSE RATIO-1)

9 2

z z UNAPG RCEL STRIP TRACHEAL GUINEA-PIG

> 2 osi o 2 toH 2

Ln FIGURE 12

Plots of log (dose ratio - 1) vs_. molar concentra tion of (-)- and (+)-INPEA obtained from guinea-pig atria and trachea. (-)-Isoproterenol was used as agonist. Num bers between the horizontal arrows connecting the regres sion lines are isomeric-activity-differences [(pA]_g of

(-) isomer) - (pA^g of (+) isomer)] . Slopes of the re gression lines for (-) and (+) isomers from atria are

1.02 and 0.60, respectively, and from trachea are 0.65 and 0.76, respectively. Vertical lines indicate S.E.M. and values in parentheses are the numbers of observations.

The tissue incubation time for both antagonists was 45 min. Asterisk denotes an asymmetric carbon atom in the structural formula.

76 LOG (DOSE RATIO-1) LOG (DOSE RATIO-1) .5 0 .5 2 2.0 0.5 2.0 1.0 2.5 A0O ( IOE *. 4 *5.8 ISOMER ) (♦ OF,A,0 A0F SMR* 84 .8 *5 ISOMER ) - ( ,A(0OF A O (-) IOE - .81 ■ 1 .44 -G ISOMER 5 OF ISOMER ,A2+ ) ) - ( OF,A? A0F ♦ IOE 3. 1 2 .4 *3 .7 ISOMER *5 ISOMER pA10OF <♦) ) - ( F pA,0 O A O ( SMR* 72 .7 *4 ISOMER ) (♦ 7.12 OF ISOMER* ,A, ) - ( OF ,A2 10 18) * 6

( IP R T S L A E H C A R T G I P - A E N I U G 8 NEA- G ATRIA IG -P A E IN U G )

NPEA E P IN - ) - ( ) M ( T S I N O G A T N A (8) 16) ( 6 ) 2.0 CH(OH)-CH,-NH-CH () A E P N I - ) * ( ^ 1(8) 1-4 ( ( 8 8 ) ) 2.31 ■CM. ■CH. ( ( 8 8 ) ) ) 10-4 r(8) ( 8 ) TABLE 7 Effect* of Beta-receptor Antagonists on (-)-isoproterenol-induced Increases In Guinea-pig Atrial Rate

Antagonist Control In presence of antagonist* "r_r Negative log Negative log Pre-curve nolar EOSO of Pre-curve molar ED50 of Dose Xsoner Concentration rate, beats/ (-)-isoprotere- rate, beats/ (-)-isoprotere- Ratio* ■in with 8.B.H. nol with S.E.M, ain with S.B.H, nol witb S,E,H, Ha with 8.B.N. (-)-alpranolol 3 x 10"® 133 i 5 8. 36 ft0.07 155 ft8 7.61 ft0.09 6 6 ft0.4 10~7 137 i 3 8.17 ft0.02 146 ft5 6.30 ft0.04 8 78 ft7 3 x 10"7 149 t 5 8.01 ft0.06 169 ft 5.78 ft0.04 8 181 ft 19 (♦)-alpranolol 3 x 10° 13% ft 4 8.41 ft0.05 150 1 7.79 ft 0.04 6 4 ft 0.2 io-6 137 ft 5 8.17 ft0.05 143 ft 7.17 ft 0.1 a n ft 2 10"5 159 ft 7 8.11 ft 0.06 145 ft 6.27 ft 0.07 6 73 ft 10 (-)-aotalol 10”* 15%ft 8 7.95ft0.06 159 ft 7,13 ft 0.05 8 7 ft 0.4 3 * 10** 1%% ft 4. 8.10 ft 0.07 162 ft 6.80 ft0.07 8 21 ft 2 xo’5 142 ft 4 8.17 ft 0.01 157 ft 6.42 ft 0.06 8 59 ft 7 (♦)-aotalol 3 x 10”5 153 ft 5 8.07 ft 0.05 163 ft 7.40 ft 0.05 8 5ft0.6 10” * 141 ft 3 8.13 ft 0.06 149 ft 6.98 ft 0.08 8 14 ft 1 3 x 10’* 141 ft 9 1.28 ft 0.04 141 t 6.71 ft 0.09 * 38 ft 5 (-)-IMPEA 10”* 142 ft 5 8.48 ft 0.06 164 ft 7.56 ft0.08 8 8 ft o.s 3 x 10"* 135 ft 4 8.30 ft 0.06 160ft 6.96ft0.08 8 23ft3 10-5 138 ft 3 8. 25 ft 0.07 153 ft 6.39 ft 0.06 8 77 ft 12 (+)-INPEA 10-5 138ft 4 8.55 ft 0.06 137 ft 8.09 ft 0.07 6 3 ft 0.3 3 X 10"5 132 ft 9 8.38 ft 0,08 131 ft 7.68 ft 0.09 8 5 ft 0.5 10** 144 ft 3 8.29 ft 0.04 122 ft 7.33 ft 0.03 8 9 ft 1 (I)-Praetolol® 3 x 10"7 135 ft 6 8.34 ft 0.07 142 ft 7.72 ft 0.06 8 2 ft 0.11 10** 140 ft • 8.49 ft 0.09 161 ft 7.47 ft0.1 8 6 ft0.3 3 x 10”* 138 ft 4 8.49 ft 0.06 151 ft 7.23 ft0.04 8 8 ft9.9

•incubation time, *5 min, bOose Ratio • antilog [(negative log nolar ED50 without antagonist) - (negative log noise *059 with antagonist)! Rvalues for dose ratio of praetolol oorrected for changes in sensitivity* - nuBber of observations* CO 79

TABLE B Effects of Beta Receptor Antagonists on (-)-isoproterenol-induced

Relaxation of Guinea-pig Traceal Strips

Antagonist Negative log molar ED50 of (-)-isoproterenol with S.E.M. Concentration In presence Dose Ratio*5 Isomer Control with S.E.M.

(-)-alprenolol 3 x 10"9 00 ± 0.06 7.59 ± 0.1 6 8 ± 1 10"7 8.06 i 0.09 6.10 t 0.05 8 96 1 13

3 x IO-7 7.93 ± 0.06 5.66 i 0.04 B 195 ± 18

(+)-alprenolol 3 x 10"7 8.38 ± 0.04 7.30 i 0.07 7 12 ± 1 io-6 8.06 ± 0.09 6.72 i 0.1 8 23 ± 3

3 x 10"6 7.91 ± 0.06 6.12 i 0.05 8 64 1 8 10-5 7.86 t 0.1 5.81 t 0.04 8 143 ± 43 (-)-sotalol 3 x 10-7 8.01 i 0.07 7.12 ± 0.07 6 8 ± 1 3 x 10“6 7.87 ± 0.1 6.33 ± 0.06 8 37 ± 5

IO-5 7.95 1 0.09 6.12 i 0.04 8 72 ± 9 3 x 10-5 8.04 ± 0.1 5.86 t 0.08 8 153 ± 11 (t)-sotalol 3 x 10"5 7.83 ± 0.09 6.83 t 0.08 8 10 1 1 10” “ 7.95 ± 0.06 6.70 1 0.05 8 18 ± 1

3 x 10"“ 7.93 i 0.08 6.39 ± 0.07 8 36 ± 5 (-)-INPEA IO'6 7.96 i 0.07 7.09 t 0.07 6 7 ± 0.5 IO”5 7.92 ± 0.09 6.40 i 0.06 8 35 ± 4 3 x IO-5 7.92 ± 0.03 6.07 t 0.04 8 74 ± 5

(+)-INPEA 3 x 10 "5 8.06 1 0.08 7.69 ± 0.07 8 2 ± 0.2 io'* 7.96 i 0.06 7.28 i 0.06 8 5 ± 0.3 3 x 10-U 7.93 1 0.06 6.97 ± 0.03 8 9 1 0.7 (±)-practolol0 3 x 10“6 8.22 ± 0.06 7.77 i 0.08 8 2 ± 0.1 IO"5 8.10 ± 0.07 7.38 1 0.06 8 4 ± 0.3 3 x 10"5 8.18 ± 0.1 7.13 i 0.06 8 9 1 1

^Incubation time, 1)5 min. “Dose Ratio - anti log [(negative log molar ED50 without antagonist) - (negative log molar ED50 with antagonist)] . ^Values for dose ratio of practolol corrected for changes in sensitivity. N - number of observations. in trachea, it is alprenolol>sotalol>INPEA. This change in order is due to the fact that sotalol is apparently more active in trachea than atria. For comparative purposes, results obtained with (±)-practolol and (-)- sotalol in atria and trachea are represented in Figures

13 and 14, respectively (see also Tables 7 and 8). Each blocker displays so-called "selective" antagonism, but in opposing directions. After the dose ratios are corrected for changes in sensitivity, pA2 values indicate that (-)- sotalol is 18 times more active in trachea than atria, while (±)-practolol is 8 times more active in atria than trachea. The slopes of the regression lines appear to play a more important role in determining pA2 values for

(-)-sotalol since higher concentrations produce more similar degrees of antagonism in the tissues (Figure 14).

Slopes of the regression lines are indicated in the

Figure Legends. For (+)-practolol, slopes in atria and trachea are more similar, however, after the dose ratios are corrected for changes in sensitivity, the apparent degree of "selectivity" exhibited by this blocker dimin ishes .

The slopes of the regression lines for each pair of isomers on the same tissue are reasonably close except for those of INPEA in isolated atria (Figure 12). Slope values obtained with all antagonists are smaller in trachea than in atria. The potency of (+)-INPEA is low and the FIGURE 13

Plots of log (dose ratio - 1) vs_. molar concentra tion of (±)-practolol obtained from guinea-pig atria and trachea. (-)-Isoproterenol was used as agonist. Antagon- ist-incubation time in both tissues was 45 min. Corrected values were calculated as described in Methods. Slopes of the regression lines for corrected and uncorrected values from atria are 0.99 and 0.73, respectively, and from trachea are 0.83 and 0.75, respectively. Vertical lines indicate S.E.M. and each point represents an average of 8 observations. The difference in corrected pA2 values between tissues is 0.89 log units or 8 fold.

Asterisk denotes an asymmetric carbon atom in the struc tural formula.

81 LOG (DOSE RATIO - I ) - 2.0 0.2 5 0 UNAPG TI 0- -0 GIE-I TRACHEA GUINEA-PIG 0 - - 0 ATRIA GUINEA-PIG D - - B □ GUI APG ARA 0 ATRIA EA-PIG IN U G □ B NORCE ;p2 25 UCRETD p2 84 .8 5 pA2= UNCORRECTED; 5 .2 7 pA2= ; UNCORRECTED ORCE;p2 43 CRETD p =5. 4 .5 2=5 pA CORRECTED; 3 .4 6 pA2= CORRECTED; CH3 - C O - N H - ^ ) - 0 - CH2 - CH (OH) - CH2 - NH - CH - NH - CH2 - (OH) CH - CH2 - 0 - ) ^ - H N - O C - CH3 ( ± ) - P R A C T O L O L ( M ) ------NEA- G TRACHEA IG -P A E IN U G 0 CH CH CO to FIGURE 14

Plots of log (dose ratio - 1) vs. molar concentra tion of (-)-sotalol obtained from guinea-pig atria and trachea. Corrected values were calculated as described in Methods. Slopes of the regression lines for corrected and uncorrected values from atria are 1.12 and 0.99, respectively, and from trachea are 0.6 8 and 0.65, respec tively. Vertical lines indicate S.E.M. and the number of observations are the same as in Figure 11. The difference in corrected pA2 values between tissues is

1.25 log units or 18 fold.

83 LOG (DOSE RATIO-I) 0.5 2.0 2.5 10 ' 8 GUI PI ATRIA IG -P A E IN U G D 0 0 G U IN E A -P IG ATRIA ATRIA IG -P A E IN U G 0 NORCE , A2 77 ■, .7 2=6 UNCORRECTED pA CORRECTED ;p A 2= 6.21 2= A ;p CORRECTED ( - ) - S O T A L O L ( M ) 0 ------O GUINEA-PIG TRACHEA TRACHEA GUINEA-PIG O UNAPG TRACHEA GUINEA-PIG 0 NORCE; A2 73 .7 7 2= pA UNCORRECTED; -p CO 85 dose ratio obtained with the lower concentration (10”^ M) is not different from sensitivity changes observed in control experiments. In addition, (+)-INPEA produced greater depression of atrial rates than other blockers

(Table 7). Thus, the slope drawn for this agent in atria may not represent its true regression. However, the individual values still give some indication of the potency of (+)-INPEA relative to its (-) form.

Non-specific effects of (+)-INPEA were also noted in guinea-pig tracheal strips. The high concentration,

3 X 10"^ M, reduced the degree of carbachol-induced tone by less than 50 percent of the control response. The result was a change in slope of the treated dose-response curve giving a non-parallel shift. This reduction in tone was corrected by constructing the second dose-response curve in the presence of a higher concentration (10”^ M) of carbachol. Responses to carbachol were not as dras tically altered by lower concentrations of (+)-INPEA and it was not necessary to induce greater tone in these experiments.

The other beta receptor antagonists reduced carbachol- induced tracheal tone to various degrees, but this effect was less than 10 percent of the maximal tension changes.

These effects of the blockers did not alter the parallel nature of the dose-response curves of the agonist. Changes

of resting tracheal tension were not quantitated since 86

tension was maintained at 5 g throughout drug contact

periods.

The beta receptor blockers produced various degrees

of changes in basal atrial rate. Results are summarized

in Table 7. Since the drug-induced rate effects cannot be accurately determined in the presence of the decelera

ting actions of phentolamine, the initial rates before

each dose-response curve can only be compared with those

obtained from separate control experiments (Table 3).

In many cases, including the control experiments, initial

atrial rates were higher before the second dose-response

curves to the agonist. The reasons for this are not clear,

but the higher rates may result from incomplete washout of

the agonist. In some instances, the beta receptor anta

gonist may increase atrial rate through its own intrinsic

actions (Ariens, 1967). Unless these rates were much

higher than those before the first dose-response curves

(30 to 40 beats per min), the parallel nature of the shift

was not altered. In cases where a non-parallel shift of

the treated dose-response curve was obtained, the results

were discarded.

Combinations of isomers of beta receptor antagonists

Conceivably, activity differences between a given

pair of antagonists could reflect the actions of one at

sites different from the other. In order to test whether 87

the isomers are acting at the same sites, isomer combina

tion studies were performed on guinea-pig atria. If the

antimers compete for the same site, the dose ratio for their combination (DR]_+2) will be equal to DR}_ + DR2 - 1.

If they act at different loci, DR ]_+ 2 = DRj• DR2 (Paton and

Rang, 1965). Figure 15 illustrates the dose-response

curves for (-)-isoproterenol from guinea-pig atria

obtained in the absence and presence of (-)-alprenolol.

3 X 10”9 M; (+)-alprenolol, 3 X 10”^ M and the combination

of these concentrations of each isomer. When combined,

the antagonists were added one right after the other and both remained in contact with the tissue for 45 min.

Table 9 depicts data taken from these curves as well as

from similar curves obtained with the other two antagonists

used in this study. The results from all experiments are

consistent with those predicted from the equation for

competition.

Rate of onset of beta receptor blockade

As outlined by Furchgott (1967), one of the criteria

to be satisfied when using competitive antagonists is that

sufficient time be allowed for equilibrium to be established.

Adrenergic-antagonism develops slowly and in some cases,

high concentrations attain a maximum and fade to a new

equilibrium level (Paton, 1967). In general, most anta

gonists achieve maximum effects more rapidly at higher FIGURE 15

Log-dose-response curves for (-)-isoproterenol from guinea-pig atria in the absence and presence of (-)-,

(+)- and (-)- and (+)-alprenolol. Numbers between the horizontal arrows connecting the curves are dose ratios.

When combined, antagonists were added one right after the other and remained in contact with the tissue for 45 min. Vertical lines indicate S.E.M. and each curve represents 6 observations. The theoretical value for competition of the two agents for the same site is 9, while that for action at different sites is 24 (Paton and Rang, 1965).

88 V. POSITIVE CHRONOTROPIC RESPONSE

il f i | s,

o, o>

68 TABLE 9

Effects of Combinations of Optical Isomers of Beta Receptor Antagonists

on Dose Ratios Obtained on Guinea-pig Atria

Observed Antagonist dose-ratio Theoretical® dose ratio Dose Ratio (DR) a Nb for N* after combination Concentration with S.E .M. combination0 For For No Isomer (Molar) with S.E.M. Competitionf Competition^ 00 i—i +i

(-)-alprenolol 3 x 10"9 6 ± 0.4 6 6 9 24 (+)-alprenolol 3 x IO-7 4 ± 0.2 6 VO 1—1 1 o

(-)-sotalol i 7 ± 0.4 8 11 + 0.5 4 11 35 (+)-sotalol 3 x 10“5 5 ± 0.6 8

(- ) -INPEA 10"6 _ 8 + 0.5 8 11 ± 2 4 12 40 (+)-INPEA 3 x IO"5 5 ± 0.5 8

aDose Ratio = antilog [(negative log molar ED50 without antagonist) - (negative log molar ED50 with antagonist)] . = number of observations for single isomers of antagonists. cCombination of isomers = number of observations for combined isomers of antagonists. eAfter Paton and Rang (1965). f°Rl+2 = DR1 + dr2 " 1

g°Rl+2 = DR1 *DR2

\D O 91 concentrations (Paton, 1961). Even though 45 min is consi dered sufficient time for full development of blockade by most beta receptor antagonists (Mclnerny et al., 1965), the influence of concentration on the rate of development of blockade has not been described. Furthermore, many studies have utilized short times of antagonist incubation in studies with "selective" antagonists (e.g., Takagi and

Takayanagi, 1970). In order to define the role of time of antagonist-tissue contact time and concentration, the rate of onset of beta receptor blockade was studied. To achieve a better separation of antagonist effects at the various time intervals, concentrations which produce large dose ratios must be used. Since all (+) isomers are less potent than the corresponding (-) forms, they must be applied in higher concentrations for equal effects.

Therefore, nonspecific effects of the (+) isomers must also be considered in selecting the appropriate antagonist for rate studies. The isomers of alprenolol satisfy these criteria better than the other available antagonists and were therefore used in these experiments. From the data obtained at 45 min incubations, equiactive concentrations of 10“7 m . for (-)-alprenolol and 10"^ Mi for (+)-alprenolol were selected. Equimolar concentrations (10"5 m ) were compared in the same studies.

Figures 16 and 17 illustrate results obtained at the 92 several time intervals of antagonist contact. In Figure

16, the log (dose ratio) values for equiactive concentra tions of the isomers are plotted. Table 10 contains the data used to construct these curves. To show the data from equimolar concentrations in perspective, all values were converted to a percent of the average log (dose ratio) obtained after 120 min incubations and displayed in Figure

17 and Table 11.

Several features are immediately obvious:

1) The antagonist equilibration profiles in the two tissues are different. Greater degrees of antagonism are attained faster in guinea-pig trachea.

2) Concentrations of the isomers equiactive at 45 min are not equally active after shorter times of incuba tion. The consequences would be smaller isomeric-activity- differences in atria than trachea at short time intervals of incubation.

3) Equimolar concentrations of the isomers do not differ markedly in their equilibration patterns in either tissue. Therefore, the processes involved in allowing access of each isomer to the receptors appear similar in the same tissue. FIGURE 16

Plots of log (dose ratio) vs. time of incubation with equiactive concentrations of the isomers of alpre nolol from guinea-pig atria and trachea strips. Log

(dose ratio) values were calculated by the formula:

[(negative log molar ED50 without antagonist) - (nega tive log molar ED50 with antagonist)] . Vertical lines indicate S.E.M. and n = number of observations for the several time intervals (see also Table 10). Tracheal strips were exposed to phentolamine, 10“5 M, 10 min prior to saline or antagonist. (-)-Isoproterenol was used as agonist. For further details, see appendix

Tables 13 and 14.

93 log (dose-ratio) 2.5 2.0 1.5 unapg atria Guinea-pig 25 n = 4 to 4 =6 56 120 65 45 ie f nuain (min) incubation of Time unapg rcel strip trocheal Guinea-pig n = 3to = 8 25 —O( -lrnll 0 M 5 10- )-alprenolol, O(+ O— —©()apeoo, 07M I0"7 ©(—)-alprenolol, ©— 45 65 120

-tr VO TABLE 10

Effects of Time of Antagonist Incubation on Blocking Potency of Isomers

of Alprenolol in Guinea-pig Atria and Trachea

Isolated guinea-pig Isolated guinea-pig right atria tracheal stripe

Time of iog (dose ratio)!: S.E.M. log (dose ratio)a ± S.E.M. antagonist (-)-alpre- (-)-alpre (+)-alpre (-)-alpre (-)-alpre (+)-alpre incubation nolol nolol nolol Pb nolol nolol nolol Pb (min) 10"5M 10~'M 1 0 "5m value 10 M 1 0 “7M 10 “5M value

3 1.15 ± 0.08 (6 ) c

1 0 3.09 i 0.09 1.07 i 0.02 1.48 ± 0.05d < . 0 0 1 2 . 6 8 4 0 . 1 1.51 ± 0.03 1.73 ± 0.07 <.05 (6 ) (6 ) (6 ) (4) (3) (4)

25 3.05 i 0.09 1.58 i 0.06d 1.82 ± 0.04d < . 0 1 2.90 4 0.08 1.69 4 0.08 1.96 4 0.09 N.S. (6 ) (6 ) (6 ) (6 ) (5) (5)

45 3.43 ± 0.06d 1.84 ± 0.07d 2 . 0 1 ± 0.08 N.S. 3.20 ± 0.05d 1.96 4 0.05d 2.05 ± 0.1 N.S. (6 ) (5) (4) (4) (8 ) (8 )

65 3.59 ± 0.08 2.07 ± 0.1 2.09 ± 0.05 3.56 4 0.03d 2.02 4 0.16 2.12 4 0.12 (6 ) (6 ) (6 ) N.S. (4) (4) (4) N.S.

1 2 0 3.57 ± 0.1 2.33 ± 0.12 2.04 ± 0.1 3.47 ± 0.04 2.15 ± 0.15 2.25 i 0.14 (5) (4) (6 ) N.S. (4) (4) (4) N.S.

aLog (dose ratio) = (negative log molar ED50 without antagonist)- (negative log molar ED50 with antagonist). “Significance of log (dose ratio) of (-)-alprenolol, 10"7M yg. log (dose ratio) of (+)-alprenolol, 10_5M. cValues in parenthesis » numbers of observations. ^Indicates significant difference (p <.05) from log (dose ratio) value obtained at previous incubation time for same antagonist and concentration. tracheal strips were exposed to phentolamine, 10-5M, 10 min prior to Baline and antagonist. FIGURE 17

Plots of percent of log (dose ratio) at 120 min vs. time of incubation with isomers of alprenolol from guinea-pig atria and tracheal strips. Percent of log

(dose ratio) was calculated by the formulas log (dose ratio) at specified time/average log (dose ratio) at

120 min X 100. Vertical lines indicate S.E.M. and n = number of observations for the several time intervals

(see also Table 11). Tracheal strips were exposed to phentolamine, 10”^ m , 10 min prior to saline or anta gonist. (-)-Isoproterenol was used as agonist. For further details, see appendix Tables 13 to 15.

96 Guinea-pig atria Guinea-pig tracheal strip

100 o 0J

.2-80

n= 4 to 6 n = 3 to 8

40 li 1 « i I i t i I i i i I I I i l l I. \ 1 .1 l - l - i___ 3 10 25 45 65 10 25 45 65 Time of incubation (min)

ID TABLE 11

Percent of Log (dose ratio) Values at Different Times of Incubation

of Isomers of Alprenolol in Guinea-pig Atria and Trachea

Isolated guinea-pig Isolated guinea-pig right atria_____ tracheal strip3 Percent of log (dose ratio)^ Percent o£ log (dose ratio)d Time of ± S.E.M. i S.E.M. antagonist (-)-alpre (-)-alpre (+)-alpre (-)-alpre (-)-alpre (+)-alpre incubation nolol nolol nolol Pb nolol nolol nolol Pb (min) 10-5M 10-7M 10*5M value 10“5M 10 ~7M 10 "5M value

3 57 ± 4 (6 ) c

8 6 < . 0 1 1 0 1 3 46 i 1 72 ± 2 77 + 3 70 i 1 70 ± 3 (6 ) (6 ) (6 ) (4) (3) (4) N.S.

25 8 6 i 3 6 8 ± 3 89 t 2 84 i 2 78 ± 4 87 ± 4 (6 )(6 )(6 ) N.S. (6 ) (5) (5) N.S.

45 96 ± 2 79 + 3 99 + 4 92 + 1 91 + 3 91+5 (6 ) (5) (4) N.S. (4) (8 ) (8 ) N.S.

65 1 0 1 ± 2 89 ± 4 103 + 2 103 ± 1 94 ± 7 94 ± 6 (6 )(6 )(6 ) N.S. (4) (4) (4) N.S.

1 2 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 (5) (4) (6 ) (4) (4) (4)

aPercent of log (dose ratio) = log (dose ratio) at specified time/average log (dose ratio) at 120 x 100. For log (dose ratio) values, see Table lo. "Significance of percent log (dose ratio) of (-)-alprenolol, 10”5m v s . percent log (dose ratio) of (+)-alprenolol, 10“*M. ‘Values in parenthesis = numbers of observations. ^Tracheal strips were exposed to phentolamine, 10- 5 M, 10 min prior to saline and antagonist. CHAPTER IV

DISCUSSION

If accurate dissociation constants for agonist- or antagonist-receptor complexes could be determined for single agents, they would be expected to be identical for identical receptor types. Presently, the best estimates of dissociation constants for beta receptor antagonists are only apparent (Furchgott, 1967). Since an irreversible inhibitor of beta receptors is not avail able, relative potencies have been considered the best tools for studying agonist-receptor interactions. The previous classifications of beta adrenergic receptors have been made using these criteria and are interpreted to mean that there are certain molecular dissimilarities between the receptors of the heart (3-1) and trachea

(3-2). From the concept of the use of isomeric-activity- ratios to differentiate adrenergic receptors (Patil, 1969) , it follows that the abilities of the 3-1 and 3 - 2 sites to interact with (-) and (+) isomers of agonists and anta gonists will not be the same. The only means of deter mining such interactions are through potency estimations and activity differences between the optical isomers 100 should provide results consistent with those from other criteria. In other words, isomeric-activity-differences should be different in the two tissues.

With the aid of optical isomers, the diffusion fac tors involving access to the receptors in different tissues are better controlled since each pair of isomers has identi cal physico-chemical properties. Therefore, reasonably accurate determinations of isomeric-activity-differences should be found by eliminating the processes acting to remove these agents from receptor regions or result in antagonistic actions (see Introduction). In most cases, these factors have been shown to be stereoselective and would be expected to alter observed activity ratios in a manner dependent upon their relative importance in each tissue.

Certain assumptions should be valid for the isomeric- activity-dif ference to be a useful criterion in differen tiating beta receptors. Many are the same as those dis cussed by Furchgott (1967) and Patil et al. (1970b). As they relate to the present studies, the assumptions are:

1) that the major pathways for removal of each isomer are sufficiently eliminated or controlled in each tissue.

For agents like norepinephrine and epinephrine which are actively accumulated by adrenergic nerve terminals, their relative potencies in a given tissue may depend upon the 101 importance of the neuronal uptake mechanism in relation to the observed response and the affinity of each agent for that mechanism. For example, neuronal uptake appears to play little role in the observed responses to norepine phrine in guinea-pig intestinal smooth muscle (Govier et al. , 1969) and rabbit aortic strips (Furchgott, 1967).

In most tissues, norepinephrine appears to have a higher affinity for uptake sites than epinephrine (Iversen, 1967).

The isomeric-activity-ratios for activation of alpha re ceptors by these agents are inversely related to the density of adrenergic innervation in the several tissues

(Patil et al., 1970b). After cocaine, this relationship is abolished due to greater potentiation of responses to the (-) isomers. According to Trendelenburg (1969), (-)- and (+)-norepinephrine may be neuronally accumulated to equal extents in most tissues, but in the presence of cocaine, the activity of the (+) isomer is not greatly altered since it produces its pharmacological effects only at concentrations which saturate the uptake sites. The apparent stereoselectivity of COMT for isoproterenol isomers observed in the present studies may be explained in a similar manner (Garg et al., 1970). The lack of effect of tropolone on positive chronotropic responses to norepinephrine and epinephrine may reflect substrate specificities of COMT or incomplete inhibition of uptake 102 by cocaine. Since there is no way to determine the effec tiveness of cocaine and tropolone in each experiment, iso meric- activity-dif ferences may be slightly altered by incomplete actions of these compounds. Their concentra tions were selected on the basis of those found to be maximally effective in other tissues (Iversen, 1967;

Kalsner and Nickerson, 1969). Extraneuronal uptake or

"uptake2 " (Iversen, 1967) does not appear to be stereo selective (Patil and Jacobowitz, 1968; Gillespie et al.,

1970) and should not alter observed activity ratios. The enzyme monoamine oxidase (MAO) should have no influence since isoproterenol is not a substrate for deamination and epinephrine and norepinephrine are prevented access to this predominantly intraneuronal enzyme by the action of cocaine.

2) that antagonistically acting alpha receptors do not

contribute to any of the observed responses. The concen tration of phentolamine utilized in these experiments is

about 10 times higher than its pA^g (Hill and Kohli, 1967)

and is expected to combine with 99 percent of the available

alpha receptors (Paton, 1961).

3) that the effects of cocaine, tropolone and phen tolamine are limited to those actions for which they were

specifically used. If they have other actions which

influence responses to the isomers, these should not be stereoselective. It has been suggested that cocaine 103 modifies alpha adrenergic receptors (Maxwell et al., 1968;

Reiffenstein, 1968). If this is true, cocaine appears to alter the alpha receptors in several tissues to the same extent (Patil et al., 1970b).

4) that both isomers of the same agonist or anta gonist produce all of their effects by attaching to the same specific receptor site. On the basis of combined antagonism, this appears to be true for the antagonists used in this study. The inability of (+)-norepinephrine to produce maximum responses may indicate a non-specific action for this isomer (Draskoczy and Trendelenburg, 1968).

However, this effect appears to be slight and it is assumed that elimination of this activity would allow maximum re sponses to be obtained with this agent. Since agonists which interact with identical receptors will be equally antagonized by the same blocking drug (Arunlakshana and

Schild, 1959), determination of pA 2 values for a beta receptor antagonist against (-) and (+) isomers of a given agonist should provide better information about the

identity of the sites activated by these compounds.

5) that agonists and antagonists interact with

similar molecular configurations of the receptor site.

This assumption is necessary for valid use of antagonists.

It appears highly probable that interactions of isopro

terenol and the beta blockers are at similar points on the 10 4 receptor since their chemical structures are very similar, many of the antagonists induce responses characteristic of beta receptor activation and are more specific in anta gonizing beta receptors than other receptor antagonists

(Ariens, 1967; Patil et a_l. , 1968).

6) that the same combining ratio of drug molecule- receptor interaction occurs for each isomer in both tissues. For purposes of this experiment, it is not necessary to assume that this drug-receptor combination ratio is one. Wenke et al. (1967) have porposed that this relationship may vary depending upon the tissue and beta receptor agonist or antagonist utilized. Thus, bi- and tri-molecular interactions could occur. It is possible that the low slope values for antagonists obtained from trachea in the present experiments are due to different ratios of drug combinations (Levy and Wilkenfeld, 1970) , but they may also be related to the degree of tone induced in the preparation (Patil, 19 68) or other factors involving agonist distribution (R.F. Furchgott, personal communica tion) .

7) that events between receptor activation and pro duction of the response do not affect the isomeric-activity- differences. Sutherland et al. (1968) and Robison et al.

(1970) have discussed the possibilities that adenyl cyclase is the beta receptor responsible for tissue responses to 105 catecholamines. A simplified scheme of events proposed to occur in the process is:

catecholamine 1 > ATP 5 '-AMP, adenyl \ phospho cyclase diesterase Cl ~ niuin 1 response

Agonist-induced activation of adenyl cyclase would lead to accumulation of cyclic AMP, the "second messenger" responsible for bringing about a response in the tissue.

Theophylline is thought to potentiate responses to beta receptor agonists by inhibiting phosphodiesterase, there by allowing a greater accumulation of cyclic AMP (Butcher and Sutherland, 1962; Rail and West, 1962; Wilkenfeld and

Levy, 1968). Positive chronotropic effects of theophyl

line may be related to this action. Since isoproterenol- induced responses were not stereoselectively altered by theophylline, the initial assumption (No. 7) would appear valid for this one step.

8) that 45 minutes is sufficient time for equilibra tion of all concentrations of both isomers of the anta gonists in both tissues and that both isomers come to

equilibrium via the same process (i.e., simple diffusion).

Studies on the rate of onset of beta blockade appear to validate this assumption for the isomers of alprenolol. 106

Equilibration appears to be more rapid in trachea and this may reflect the relative thinness of this tissue.

9) that the beta receptor antagonists used in this study are not metablized or distributed stereoselectively in either tissue during incubation periods. These com pounds are not substrates for COMT or MAO and the extent to which other enzyme systems which metabolize them exist in isolated tissues is probably small. Since antagonism is not reduced after 120 min incubation periods, any such effects appear unimportant.

10) that isomeric-activity-differences are not altered by the experimental procedures used in the two tissues.

Studies of beta receptors in atria and trachea are neces sarily different and comparisons are difficult since experimental factors may obscure pharmacological evalua tion of drug potencies. Induction of tone in trachea introduces physiological antagonism and membrane depolari zation accompanied by tissue contraction may alter the ionic environment of the receptors. An analogous situa tion may exist in atria when rates are lowered by phentolamine and differences in baseline between first and second curves may change the dose-response profile. With the use of optical isomers, these factors appear controlled in most cases.

11) that the responses observed with the (+) isomers

are not entirely due to contamination by their (-) forms. 107

There is no way to directly determine the optical purity of a single sample without a pure standard. It is assumed that, after repeated recrystallization, maximum purity of the (+) isomer is indicated by minimum alteration of bio logical activity (Lands et al., 1954). However, in determining isomeric-activity-differences of adrenergic compounds, it is not absolutely necessary to have an absolutely pure (+) isomer. The assumption is that the

(+) isomers do not have zero potency. Therefore, as long as the same chemical samples are used in all experiments, slight contamination by the (-) isomer can be tolerated.

Indirect evidence from two sources suggests that the (+) isomers have intrinsic activity. First, tropolone produced stereoselective potentiation of the responses to isoproterenol. If the activity of the (+) isomers were solely due to the presence of small amounts of its antimer, tropolone would have potentiated responses to both (-)- and (+)-isoproterenol to equal extents. The second evidence may be presented in light of the Easson and Stedman hypothesis (1933) that (+) isomers act as if the OH group were missing (see also Patil et al., 1967).

Thus, the potencies of the (+) isomers should be the same as those of their desoxy derivatives. Figure 18 illustrates dose-response curves to (+)-isoproterenol and desoxy-isoproterenol obtained from guinea-pig right FIGURE 18

Log-dose-response curves for (+)-isoproterenol and desoxy-isoproterenol obtained from atria taken from normal guinea pigs. Only one compound was tested in each tissue in the presence of tropolone, 3 X 10“5 m , added 30 min previously. Only one drug was tested on each tissue since effects of the desoxy derivative could not be washed away. Negative log molar ED50 values for

(+)-isoproterenol and desoxy-isoproterenol are 5.77

(S.E.M. + 0.09) and 5.82 (S.E.M. ± 0.05), respectively.

Vertical lines indicate S.E.M. j n = number of observa tions for each curve.

108 Guinea-pig atria with Tropolone, 3xlO- 5 M a. 100

& 80

a. 40 O— O (+)- isoproterenol O— O desoxy-isoproterenol 20 n = 6

Agonist (M ) 109 110 atria in the presence of tropolone. The activities of

(+)-isoproterenol and desoxy-isoproterenol are identical.

Similar results were obtained from guinea-pig atria by

J.R. Blinks (personal communication). Since, with respect to receptor interaction, the functional groups of (+) isomers and their desoxy derivatives are similarly oriented, it is highly probable that the effects of the (+) isomers are genuine and not due to contamination by the (-) forms.

Provided these assumptions are satisfied, the avail able evidence suggests that the isomeric-activity-difference for a given pair of isomers is the same in guinea-pig atria and trachea. Small differences may be attributed to experimental variables involving one or more of the above mentioned assumptions. The best evidence from the agonists is obtained with the use of the isomers of isoproterenol where fewer experimental variables need to be controlled.

Differences in their ratios between the two tissues are

1.5 fold. Thus, it appears that the beta receptors of guinea-pig atria and trachea possess identical abilities to interact with their agonists and antagonists. It follows that the receptor sites in these two tissues are similar.

The data from the (-) isomers of isoproterenol, nore pinephrine and epinephrine can easily be arranged according to order of potencies in the two tissues (Figure 4). This Ill procedure is frequently done to subclassify beta recep tors. However, it does not appear to be sensitive enough for an accurate comparison of receptor conformations.

Similar arrangement of the (-) forms of the anta gonists used in this study reveal the apparent "selectivity" of sotalol for beta receptors in trachea. A comparison of the pA 2 values of (-)-sotalol in atria and trachea indi cates that they differ by 10 fold. According to the criterion used by others, this "selectivity" implies that different beta receptor types are present in the two tissues. However, the different pA 2 values for sotalol are obviously related to the slopes of the regression lines.

Inclusion of pA2 values of (+)-sotalol in the comparisons provides an internal control and illustrates the erroneous conclusions that can be made when only single agents are investigated. The isomeric-activity-difference for sotalol (1.7 log units) is the same from atria and trachea.

Practolol has been previously considered a valuable compound in supporting the existence of 3-1 and 3-2 adrenergic receptors. Results from the present experi ments indicate that the difference in its pA 2 values between atria and trachea is less than 1 log unit (10 fold).

Although slopes of the regression lines do not appear to influence pA 2 determinations for practolol, such small differences in pA 2 values between tissues are of question able significance (Kohli, 1969). Recent experiments by 112

Levy and Wilkenfeld (1970) attribute larger pA 2 values to practolol in guinea-pig tracheal strips after 2 hr contact with the tissue. Their value of 7.3 is about 100 times that reported in this study for 45 min incubation periods in the same tissue. For this drug, the time of incubation required to achieve equilibrium in trachea may be greater than for other antagonists and this factor must be ad justed accordingly when investigating its blocking pro perties. Testing the optical isomers of practolol may provide better information about its "selectivity" for the so-called $-1 receptor.

Another interesting observation from the present study is the apparent lack of relationship between potency and the isomeric-activity-difference for agonists and antagonists. If isomeric-activity-ratios indicate recep tor conformations, these values are expected to increase with increasing racemic potency (Pfeiffer, 1956). In other words, if the more potent isomers attach to the receptor surface with a "tighter fit", small differences in structure, such as changes in configuration about an asymmetric carbon atom, should reduce the activities of the less potent isomers to a greater extent than if the compound were less potent. To a large degree, this relationship depends upon the location of asymmetry in the drug molecule. If it is in a "non-critical" moiety for 113 binding, its influence will be less since its contribution to the drug-receptor complex is small (Ariens and Simonis,

1968). The alcoholic function of beta receptor agonists and antagonists is considered a "critical" portion of the molecule since, without it, activity is appreciably re duced (Ariens, 1967; Patil, 1968; Patil et al., 1970a; present investigations). Accordingly, the more potent agonist, isoproterenol, has a larger ratio of activity between its optical isomers. However, no good correlation exists for the isomers of epinephrine and norepinephrine.

Furthermore, one of the so-called "selective" agonists, soterenol, is about 10 times more potent than isoproterenol in trachea, but has an isomeric-activity-ratio of about

100 (R.J. Seidehamel, personal communication).

Similarly, the antagonists used in this study display various potency values, but all of them have an isomeric- activity-dif ference of about 2 log units. According to

Pfeiffer's relationship, the isomers of alprenolol should have greater activity differences than the other antagonists

since it is apparently the more potent beta receptor blocking agent. This lack of relationship may be ex plained if the (+) isomers of the antagonists have no beta

receptor affinities and their observed effects are due to

their contaminating (-) forms. However, as shown by Patil

(1968), desoxy-sotalol has similar potency to (+)-sotalol. 114

Thus, the previous argument for (+)-isoproterenol activity based on the Easson-Stedman hypothesis appears equally valid for the antagonists.

These observations support the contention that relative potencies of the various compounds are not only indicative of their abilities to combine with the receptor, but also reflect factors which influence their access to the active sites. The ED50 and pAx values of single agents may fall anywhere along the log-dose axis and not accurately reveal their receptor combining abilities. When optical isomers

are compared under proper experimental conditions, the factors which result in such variability appear controlled and the important drug parameter becomes the isomeric-

activity-dif ference. Thus, the results indicate that, when able to be determined, the ultimate beta receptor combining abilities of alprenolol, sotalol and INPEA will be identical and the true order of affinities for the agonists will be isoproterenol>norepinephrine>epinephrine in both guinea-pig atria and trachea.

The physico-chemical properties which determine beta receptor drug actions are not well understood. There

appears to be no relationship between lipid solubility or pK and beta receptor blocking activity (Levy, 1968). For example, the compound Ko 592 has greater blocking activity

in rabbit atria and much lower lipid solubility than 115 propranolol. Sotalol, with 150 to 200 times less potency on the same tissue, has the same chloroform:water parti tion coefficient as Ko 592. This cannot be explained on the basis of pK values since sotalol is equally basic with propranolol. The dilemma involves the present lack of knowledge about the diffusive barriers in the tissues.

In other words, are structure-activity and physico-chemical- activity relationships related to receptor of diffusion requirements ?

The conclusion that similar beta adrenergic receptors are present in guinea-pig atria and trachea does not allow extrapolation to other tissues or species. It does indi cate that results obtained with single agents should not be used as bases for conclusions regarding the receptor proper. It is possible that, like cnzymes-isoenzymes, receptors-isoreceptors may exist. In some cases, the

"selectivity" exhibited by the several agents may be a reflection of this possibility. Furthermore, the thera peutic use of these agents is very valuable, even disre garding receptor types. However, the reasons for such

"selective" actions in guinea-pig atria vs. trachea must be sought elsewhere. The present results indicate that beta receptors in guinea-pig atria and trachea may be of a single type. CHAPTER V

SUMMARY AND CONCLUSIONS

1) Under proper experimental conditions, activity

differences between optical isomers of several beta

adrenergic receptor agonists and antagonists have been

determined on guinea-pig atria and trachea in view of the

hypothesis that these tissues contain different sub

species of beta adrenergic receptor.

2) Isomeric-activity-differences for isoproterenol,

norepinephrine and epinephrine from tissues taken from

reserpine-pretreated guinea pigs are, for atria, 3.01,

2.70 and 1.80, respectively and for trachea, 2.69, 2.30

and 1.46, respectively. Thus, there is a maximum of a

2.5 fold (0.4 log unit) difference in ratio between tissues.

Where determined, these values were not different when

tissues were taken from normal animals.

3) Tropolone potentiated responses to the isomers

of isoproterenol in both tissues, stereoselectively in

favor of the (-) form. Although, in the absence of tro

polone, isomeric-activity-differences were decreased, the

same degree of reduction was observed in each tissue. The

activities of (-)- and (+)-norepinephrine and (-)- and (+)-

epinephrine were not altered by tropolone in guinea-pig

116 117 atria.

4) Theophylline potentiated responses to both isomers of isoproterenol in guinea-pig atria to the same extent in the presence of tropolone. Isomeric-activity-differences were left unaltered by theophylline.

5) The negative log molar ED50 values of (+)-isopro terenol and desoxy-isoproterenol were identical in guinea- pig atria. On the basis of the Easson-Stedman hypothesis, these results may be interpreted to mean that the activity of (+)-isoproterenol is not zero.

6) Orders of potencies of the (-) isomers of the agonists in atria were isoproterenol>norepinephrine^epinephrine. In trachea, the orders were isoproterenol>epinephrine>norepinephrine.

7) Isomeric-activity-differences for alprenolol, sotalol and INPEA in atria are 2.16, 1.68 and 2.00, respec tively, and in trachea are 1.77, 1.84 and 2.31, respec tively. There is a maximum of a 2.5 fold (0.4 log unit) difference in ratio between the tissues.

8) A comparison of the blocking activities of (-)- sotalol and (±)-practolol in guinea-pig atria and trachea illustrates that apparent "selectivity" of the former (18 times more active in trachea) may result from different pA regression coefficients which are always lower in trachea. The degree of "selectivity" exhibited by (±)- practolol (8 times more active in atria) is less under the 118

same experimental conditions. Since the isomeric-activity-

dif ferences of sotalol are the same in the two tissues,

it appears that at least 10 fold variations in activity may occur in atria and trachea without altering activity

ratios between optical isomers.

9) Time of antagonist incubation may influence

isomeric-activity-differences when determined at brief

contact periods (<45 min in atria and <25 min in trachea).

Equimolar concentrations of the isomers of alprenolol

have similar equilibration patterns in the same tissue

indicating identical means of access to the active sites.

10) Orders of potencies of the (-) forms of the

antagonists in atria were alprenolol>INPEA^sotalol. In

trachea, the orders were alprenolol>sotalol>INPEA.

11) Although, for several possible reasons, the

agonists or antagonists may occupy different positions

along the log-dose axis in the two tissues, the similarity

of the isomeric-activity-differences of each pair from

both tissues indicates that beta adrenergic receptors of

guinea-pig atria and trachea may be of a single type. APPENDIXES 120

TABLE 12

Chemicals used in the Present Study

Compounda Batch No. F.W. Source

(-)-alprenolol B 10 F 286 Dr. B. Ablad AB Hassle (+)-alprenolol B 10 F 416. . . Goteborg, Sweden

(-)-INPEA 4 1° 261 Dr. W. Murmann Selvi 6 Co. (+)-INPEA 4 1° 261. . . Milano, Italy

(-)-sotalol 3MCA347 309 Dr. W.T. Comer Mead Johnson (+)-sotalol 3MCA 346 309. . . Evansville, Ind.

(-)-isoproterenol Tu—L— 17b 397

(+)-isoproterenol R-032-SK 361 desoxy- isoproterenol Tu-I-92 2 32 Dr. S. Archer (+)-norepinephrine R-007-ZB 319 Sterling-Winthrop Research Institute (+)-epinephrine R-033-BB 332. Rensselaer, N.Y.

(-)-norepinephrine RN261(L536) 169

(-) -epinephrine RD-9 183. Regis Chemicals

Tropolone T8970-2 122. Aldrich Chemicals

Dr. A.J. Plummer Ciba Phentolamine B64-R-27 377. Summit, N.J.

Carbachol C240-9 183. Aldrich Chemicals

Cocaine 340. Merck Chemicals

Aminophylline______456. Merck Chemicals aSalt forms may be found in Methods TABLE 13

Effects of (-)-alprenolol, 10~7M, at Various Time Intervals

of Contact with Guinea-pig Atria and Trachea

Time of Isolated guinea-pig Isolated guinea-pig Incubation right atria tracheal strip with (-)- Negative log molar ED50 of Negative log molar ED50 of alprenolol (-)-isoproterenol with S.E.M. Dose (-)-isoproterenol0 with S.E.M.• Dose 10~7M In the presence Ratio" In the presence Ratio" (min) Control of antagonist N a with S.E.M. Control of antagonist N a with S.E.M.

10 8.23 ± 0.06 7.16 + 0.05 6 12 ± 0.6 8.16 i 0.08 6.65 + 0.06 3 33 ± 2

25 8.45 ± 0.05 6.87 1 0.06 6 40 i 5 8.09 ± 0.08 6.40 + 0.06 5 52 t 9

45 8.40 ± 0.07 6.56 ± 0.03 5 73 + 12 8.06 ± 0.09 6.10 ± 0.05 8 96 t 13

65 8.43 i 0.08 6.37 ± 0.1 6 133 ± 30 8.29 ± 0.17 6.27 ± 0.02 4 125 ± 41 120 8.54 ± 0.04 6.21 ± 0.16 4 233 ± 48. 8.29 ± 0.27 6.14 + 0.13 4 167 ± 51

aN = number of observations fc*Dose ratio *• antilog [(negative log molar ED50 without antagonist)- (negative log molar ED50 with antagonist)] . cObtained m the presenct of phentolamine, 10-5M, added 10 min prior to saline and antagonist. TABLE 14

Effects of (+)-alprenolol, 10~5M, at Various Time Intervals

of Contact with Guinea-pig Atria and Trachea

Time of Isolated guinea-pig Isolated guinea-pig Incubation right atria tracheal strip with (+)- Negative log molar ED50 of Negative log molar EC50 of alprenolol (-)-isoproterenol with S.E.M. Dose (-)-isoproterenol0 with S.E.M. Dose 10-5M In the presence Ratio*5 In the presence Ratio13 (min) Control of antagonist N a with S.E.M. Control of antagonist N a with S.E.M.

3 8.28 ± 0.09 7.13 ± 0.05 6 16 ± 3 10 8.28 ± 0.1 6.81 ± 0.09 6 31 ± 3 8.09 + 0.03 6.36 ± 0.05 4 56 ± 9

25 8.44 ± 0.06 6.63 ± 0.07 6 67 ± 6 8.04 + 0.13 6.09 ± 0.07 5 98 ± 18

45 8.56 1 0.08 6.54 ± 0.09 4 108 ± 20 7.86 ± 0.1 5.81 + 0.04 8 143 ± 43

65 8.50 ± 0.03 6.41 ± 0.07 6 128 ± 14 8.27 ± 0.2 6.16 ± 0.08 4 145 ± 32

120 8.20 i 0.06 6.17 t 0.04 6 122 i 24 8.15 + 0.35 5.90 + 0.25 4 212 i 76

3N = number of observations bDose ratio * antilog {(negative log molar EO50 without antagonist)- (negative log molar ED50 with antagonist)] . cObtained in the presence of phentolomine, 10”5m , added 10 min prior to saline and antagonist. TABLE 15

Effects of (-)-alprenolol, 10~5M, at Various Time Intervals

of Contact with Guinea-pig Atria and Trachea

Time of Isolated guinea-pig Isolated guinea-pig Incubation ______right atria______tracheal strip_____ with (-)- Negative log molar ED50 of Negative log molar ED50 of alprenolol (-)-isoproterenol with S.E.M. Dose (-)-isoproterenol0 with S.E.M. Dose 10“5m In the presence Ratiob In the presence Ratio*5 (min) Control of antagonist Na with S.E.M. Control of antagonist Na with S.E.M.

10 8.18 ± 0.05 5.09 ± 0.06 6 1355 ± 244 7.70 ± 0.05 5.02 ± 0.07 4 511 ± 101

25 8.09 + 0.05 5.04 i 0.04 6 1213 ± 192 7.69 + 0.1 4.78 + 0.03 6 867 ± 165 45 8.23 ± 0.07 4.80 ± 0.05 6 2846 + 450 7.78 + 0.04 4.58 + 0.02 4 1615 ± 189

65 8.33 + 0.04 4.75 t 0.08 6 4164 ± 748 8.34 ± 0.06 4.78 + 0.04 4 3681 ± 292

120 8.07 i 0.05 4.50 ± 0.1 5 4160 + 875 8.10 + 0.05 4.63 + 0.06 4 2974 ± 276

*N = number of observations bDose ratio ■ antilog [(negative log molar ED50 without antagonist)- (negative log molar ED50 with antagonist!] . CQbtained in the presence of phentolamine, 10"5M, added 10 min prior to saline and antagonist. 123 BIBLIOGRAPHY

Ahlquist, R.P.: A study of the adrenotropic receptors. Am. J. Physiol. 153: 586-600, 1948.

Ahlquist, R.P. and Levy, B.: Adrenergic receptive mecha nism of canine ileum. J. Pharmacol. Exp. Ther. 127: 146-149, 1959.

Ariens, E.J., Simonis, A.M. and Van Rossum, J.M.: Drug- receptor interaction: Interaction of one or more drugs with one receptor system. ]Di Molecular Pharmacology (E.J. Ariens, Ed.), Vol 1, 119-286, Academic Press, New York, 196 4.

Ariens, E.J.: The structure-activity relationships of beta adrenergic drugs and beta adrenergic blocking drugs. Ann. N. Y. Acad. S c i V 139: 606-631, 1967.

Ariens, E.J. and Simonis, A.M.: Chemical structure and biological activity. Some general principles. In Physico-chemical Aspects of Drug Action (E.J. Ariens, Ed.), 271-281, Pergamon Press, Oxford, 1968.

Arunlakshana, O. and Schild, H.O.: Some quantitative uses of drug antagonists. Br. J. Pharmacol. Chemother. 14: 48-58, 1959.

Barger, G. and Dale, H.H.: Chemical structure and sym pathomimetic action of amines. J. Physiol. 41: 19-59, 1910.

Barrett, A.M., Crowther, A.F., Dunlop, 0., Shanks, R.G. and Smith, L.H.: Cardio-selective S-blockade. Naunyn-Schmiedebergs Arch. Exp. Path. Pharmak. 259: 152-153, 1968.

Bergman, J., Persson, H. and Wetterlin, K.: Two new groups of selective stimulants of adrenergic 6-receptors. Experientia 2j>: 899-901, 1969.

Black, J.W. and Stephenson, J.S.: Pharmacology of a new adrenergic beta-receptor-blocking compound (Nethalide). Lancet 2: 311-314, 1962.

124 125

Black, J.W. , Crowther, A.F. , Shanks, R.G., Smith, L.II. and Dornhorst, A.C.: A new beta-receptor antagonist. Lancet 1: 1080-1081, 1964.

Brick, I., Hutchison, K.J., Roddie, I.e. and Shanks, R.G.: Cardiac adrenergic blockade by 4-(2-hydroxy-3-isopropylaminopropoxy) acetanilide (ICI 50172). Naunyn- Schmiedebergs Arch. Exp. Path. Pharmak. 259: 156- 157, 1968.

Bristow, M., Sherrod, T.R. and Green, R.D.: Analysis of beta receptor drug interactions in isolated rabbit atrium, aorta, stomach and trachea. J. Pharmacol. Exp. Ther. 171: 52-61, 1970.

Brittain, R.T., Farmer, J.B., Jack, D., Martin, L.E. and Simpson, W.T. : a- [(t-Butylamino) methyl] -4- hydroxy-m-xylene-a^otS-diol (AH 3365) : a selective 6-adrenergic stimulant. Nature 219: 862-863, 1968.

Burns, J.J., Colville, K.I., Lindsay, L.A. and Salvador, R.A.: Blockade of some metabolic effects of cate cholamines by N-isopropylmethoxamine (B.W. 61-43). J. Pharmacol. Exp. Ther. 144: 163-171, 1964.

Burns, J.J. and Lemberger, L.: N-tertiary butylmethoxamine, a specific antagonist of the metabolic actions of epinephrine. Fed. Proc. 24_: 298, 1965.

Butcher, R.W. and Sutherland, E.W.: Adenosine 3',5'- phosphate in biological materials. I. Purification and properties of cyclic 3 1,5'-nucleotide phospho diesterase and use of this enzyme to characterize adenosine 3',51-phosphate in human urine. J. Biol. Chem. 237: 1244-1250, 1962.

Cannon, W.B. and Rosenblueth, A.: Studies on conditions of activity in endocrine organs. XXIX. Sympathin E and Sympathin I. Am. J. Physiol. 10 4: 557-574, 1933.

Constantine, J.W.: The spirally cut tracheal strip pre paration. J. Pharm. Pharmacol. 3/7: 384-385, 1965.

Cullum, V.A., Farmer, J.B., Jack, D. and Levy, G.P.: Salbutamol: a new, selective 8-adrenoceptive recep tor stimulant. Br. J. Pharmacol. 35_: 141-151, 1969.

Cushny, A.R.: Biological relations of optically isomeric substances. Williams and Wilkins Co., Baltimore, 1926. 126

Dale, H.H.: On some physiological actions of ergot. J. Physiol. 34.: 163-206 , 1906.

Dixon, W.E. and Hamill, P.: The mode of action of speci fic substances with special reference to secretin. J. Physiol. .38: 314-336 , 1909.

Draskoczy, P.R. and Trendelenburg, U.: The uptake of 1- and d-norepinephrine by the isolated perfused rabbit heart in relation to the stereospecificity of the sensitizing action of cocaine. J. Pharmacol. Exp. Ther. 159.: 66-73, 1968.

Dungan, K.W. , Cho, Y.W., Gomoll, A.E., Aviado, D.M. and Lish, P.M.: Pharmacologic potency and selectivity of a new bronchodilator agent: soterenol (MJ 1992). J. Pharmacol. Exp. Ther. 164: 290-301, 1968.

Dunlop, D. and Shanks, R.G.: Selective blockade of adrenoceptive beta receptors in the heart. Br. J. Pharmacol. Chemother. 32.: 201-218, 1968.

Easson, L.H. and Stedman, E.: CLXX. Studies on the relationship between chemical constitution and physiological action. V. Molecular dissymmetry and physiological activity. Biochem. J. 27: 1257-1266, 1933.

Elliott, T.R.: The action of adrenalin. J. Physiol. 32: 401-467, 1905.

Euler, U.S.v.: A specific sympathomimetic ergone in adrenergic nerve fibers (sympathin) and its relation to adrenaline and noradrenaline. Acta physiol. Scand. 12: 73-97, 1946.

Euler, U.S.v.: Identification of the sympathomimetic ergone in adrenergic nerves of cattle (Sympathin N) with laevonoradrenaline. Acta physiol. Scand. 16.: 63-74, 1948.

Euler, U.S.v.: Noradrenaline. C.C. Thomas, Springfield, 1956.

Farmer, J.B. and Levy, G.P.: New sympathomimetic amines: actions on catecholamine receptors. Br. J. Pharmacol. 34: 698P, 1968. 127

Farmer, J.B. , Kennedy, I., Levy, G.P. and Marshall, R.J.: A Comparison of the 3-adrenoreceptor stimulant pro perties of isoprenaline, with those of orciprenaline, salbutamol, soterenol and trimetoquinol on isolated atria and trachea of the guinea-pig. J. Pharm. Pharmacol. 22:: 61-63, 1970a.

Farmer, J.B. and Levy, G.P.: Differentiation of 8-adreno- receptors by the use of blocking agents. J. Pharm. Pharmacol. 22: 145-146, 1970b.

Freund, J.E., Livermore, P.E. and Miller, I.: Manual of Experimental Statistics, pp 40-43. Prentice-Hall, Englewood Cliffs, 1960.

Furchgott, R.F.: The pharmacology of vascular smooth muscle. Pharmacol. Revs. 1_: 183-265, 1955.

Furchgott, R.F.: The receptors for epinephrine and nore pinephrine (adrenergic receptors). Pharmacol. Revs. 11: 429-441, 1959.

Furchgott, R.F.: The use of 8“haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist com plexes. In. Advances in Drug Research (N.J. Harper and A.B. Simmonds, Eds.), Vol 3, 21-55, Academic Press, London, 1966.

Furchgott, R.F.: The pharmacological differentiation of adrenergic receptors. Ann. N.Y. Acad. Sci. 139: 553-570, 1967.

Furchgott, R.F. and Bursztyn, P.: Comparison of dis sociation constants and of relative efficacies of selected agonists acting on parasympathetic recep tors. Ann. N.Y. Acad. Sci.' 144: 882-898, 1967.

Garg, B., Buckner, C., Sethi, 0., Sokoloski, T. and Patil, P.N.: Steric aspects of adrenergic drugs. XVII. Influence of tropolone on the magnitude and duration of action of catecholamine isomers. Arch. Int. Pharmacodyn. Ther. in press, 1970.

Ghouri, M.S.K. and Shibata, S.: 8-Adrenoceptor stimula ting properties of para-dimethylaminobenzaldehyde. Br. J. Pharmacol.' '39: 447-454, 1970. 12 8

Gillespie, J.S., Hamilton, D.N.H. and Ilosie, R.J.A. : The extraneuronal uptake and localization of noradrenaline in the cat spleen and the effect on this of some drugs, of cold and of denervation. J. Physiol. 206: 563- 590, 1970.

Govier, W.C., Sugrue, M.G. and Shore, P.A.: On the ina bility to produce supersensitivity to catecholamines in intestinal smooth muscle. J. Pharmacol. Exp. Ther. 165: 71-77, 1969.

Hertting, G.: The fate of H^-isoproterenol in the rat. Biochem. Pharmacol. 2J5: 18-33, 19 64.

Hill, A.J. and Kohli, J.D.: Adrenoceptive receptors in the duodenum, aorta and atria of the rabbit. J. Pharm. Pharmacol. _19: 350-354, 1967.

Holtz, P. and Palm, D.: On the pharmacology of a-methylated catecholamines and the mechanism of the anti hypertensive action of a-methyldopa. Life Sci. 6_: 1847-1857, 1967.

Hudgins, P.M. and Fleming, W.W.: A relatively nonspecific supersensitivity in aortic strips resulting from pre treatment with reserpine. J. Pharmacol. Exp. Ther. 153: 70-80, 1966.

Iversen, L.L. : The Uptake and Storage of Noradrenaline in Sympathetic Nerves. Cambridge University Press, Cambridge, 1967.

Kalsner, S. and Nickerson, M.: Disposition of norepine phrine and epinephrine in vascular tissue, determined by the technique of oil immersion. J. Pharmacol. Exp. Ther. 165: 152-165, 1969.

Kohli, J.D.: A comparative study of dopamine and noradrenaline on the rabbit aorta. Can. J. Physiol. Pharmacol. 4-7: 171-174, 1969.

Krell, R.D. and Patil, P.N.: Combinations of alpha and beta adrenergic blockers in isolated guinea-pig atria. J. Pharmacol. Exp. Ther. 170: 262-271, 1969.

Lands, A.M., Nash, V.L., McCarthy, H.M., Granger, H.R. and Dertinger, B.L.: The pharmacology of N-alkyl homo- logues of epinephrine. J. Pharmacol. Exp. Ther. 90: 110-119, 1947. 129

Lands, A.M., Luduena, F.P. and Tullar, B.F.: The pharma cologic activity of the optical isomers of isopropylarterenol (Isuprel) compared with that of the optically inactive analog l-(3,4-dihydroxyphenyl)-2- isopropylaminoethane HC1. J. Pharmacol. Exp. Ther. Ill: 469-474, 1954.

Lands, A.M.: Sympathetic receptor action. Am. J. Physiol. 169: 11-21, 1952.

Lands, A.M. and Brown, T.G.,Jr.: A comparison of the cardiac stimulating and bronchodilator actions of selected sympathomimetic amines. Proc. Soc. Exp. Biol. Med. 116: 331-333, 1964.

Lands, A.M., Groblewski, G.E. and Brown, T.G.,Jr.: Com parison of the action of isoproterenol and several related compounds on blood pressure, heart and bronchioles. Arch. Int. Pharmacodyn. Ther. 161: 68-75, 1966.

Lands, A.M., Arnold, A., McAuliff, J.P,, Luduena, F.P. and Brown, T.G.,Jr.: Differentiation of receptor systems activated by sympathomimetic amines. Nature 214: 597-598, 1967a.

Lands, A.M., Luduena, F.P. and Buzzo, H.J.: Differen tiation of receptors responsive to isoproterenol. Life Sci. 6: 2241-2249, 1967b.

Langley, J.N.: On the reaction of cells and of nerve- endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curari. J. Physiol. 3J3: 374-413, 1905.

Larsen, A.A., Gould, W.A., Roth, H.R., Comer, W.T., Uloth, R.H., Dungan, K.W. and Lish, P.M.: Sulfonanilides. II. Analogs of catecholamines. J. Med. Chem. 10: 462-472, 1967.

Levy, B.: Alterations of adrenergic responses by N-isopropylmethoxamine. J. Pharmacol. Exp. Ther. 146: 129-138, 1964.

Levy, B.: The adrenergic blocking activity of N-tert.- butylmethoxamine (Butoxamine). J. Pharmacol. Exp. Ther. 151: 413-422, 1966a. 130

Levy, B.: Dimethyl isopropylmethoxamine: A selective 3-receptor blocking agent. Br. J. Pharmacol. Chemother. 27s 277-285, 1966b.

Levy, B.: A comparison of the adrenergic receptor blocking properties of 1-(4'-methylphenyl)-2-isopropylamino- propanol-HCl and propranolol. J. Pharmacol. Exp. Ther. 156: 452-462, 1967a.

Levy, B.: Selective beta receptor blockade in rat smooth muscle. Arch. Int. Pharmacodyn. Ther. 170: 418-427, 1967b.

Levy, B. and Wilkenfeld, B.E.: The potentiation of rat uterine inhibitory responses to noradrenaline by theophylline and nitroglycerine. Br. J. Pharmacol. Chemother. 34_: 60 4-612, 1968.

Levy, B. and Wilkenfeld, B.E.: An analysis of selective beta receptor blockade. Europ. J. Pharmacol. 5: 227-234, 1969.

Levy, B. and Wilkenfeld, B.E.: The actions of selective 3-receptor antagonists on the guinea-pig trachea. Europ. J. Pharmacol. 11: 67-74, 1970.

Levy, J.V.: Myocardial and local anesthetic actions of 3-adrenergic receptor blocking drugs; relationship to physiological properties. Europ. J. Pharmacol. 2: 250-257, 1968.

Luduena, F.P., Ananenko, E., Siegmund, O.H. and Miller, L. C.: Comparative pharmacology of the optical isomers of arterenol. J. Pharmacol. Exp. Ther. 9_5: 155- 170, 1949.

Maxwell, R.A., Eckhardt, S.B. and Wastila, W.B.: Con cerning the distribution of endogenous norepine phrine in the adventitial and media-intimal layers of the rabbit aorta and the capacity of these layers to bind tritiated norepinephrine. J. Pharmacol. Exp. Ther. 161: 34-39, 1968.

Mayer, S.E., Moran, N.C. and Fain, J.: The effect of adrenergic blocking agents on some metabolic actions of catecholamines. J. Pharmacol. Exp. Ther. 134: 18-27, 1961.

Moran, N.C. and Perkins, M.E.: Adrenergic blockade of the mammalian heart by a dichloro analog of isoprotere nol. J. Pharmacol. Exp. Ther. 124: 233-237, 1958. 131

Moran, N.C.: Pharmacological characterization of adre nergic receptors. Pharmacol. Revs. _18: 503-512, 1966.

Mclnerny, T.K., Gilmour, D.P. and Blinks, J.R.: Com parison of effects of propranolol and other cardiac adrenergic blocking agents on inotropic and chrono tropic actions of catecholamines. Fed. Proc. 24: 712, 1965.

Nickerson, M.: The pharmacology of adrenergic blockade. Pharmacol. Revs. 1_: 27-101, 1949.

Patil, P.N., LaPidus, J.B. and Tye, A.: Steric aspects of adrenergic drugs. I. Comparative effects of DL isomers and desoxy derivatives. J. Pharmacol. Exp. Ther. 155: 1-12, 1967.

Patil, P.N.: Seric aspects of adrenergic drugs. VIII. Optical isomers of beta adrenergic receptor anta gonists. J. Pharmacol. Exp. Ther. 160: 308-314, 1968.

Patil, P.N. and Jacobowitz, D.: Steric aspects of adre nergic drugs. IX. Pharmacologic and histochemical studies on isomers of cobefrine (a-methylnorepinephrine). J. Pharmacol. Exp. Ther. 161: 279-295, 1968.

Patil, P.N., Tye, A., May, C., Hetey, S. and Miyagi, S.: Steric aspects of adrenergic drugs. XI. Interactions of dibenamine and beta adrenergic blockers. J. Pharmacol. Exp. Ther. 163: 309-319, 1968.

Patil, P.N.: Use of isomeric-ratio as a criterion to differentiate adrenergic receptors. J. Pharm. Pharmacol. 2/Li 628-629, 1969.

Patil, P.N., LaPidus, J.B. and Tye, A.: Steric aspects of adrenergic drugs. J. Pharm. Sci. 59: 1205-1234, 1970a.

Patil, P.N., Patel, D.G. and Krell, R. : Steric aspects of adrenergic drugs. XV. Use of isomeric-activity- ratio as a criterion to differentiate adrenergic receptors. J. Pharmacol. Exp. Ther. in press, 1970b.

Paton, W.D.M.: A theory of drug action based on the rate of drug-receptor combination. Proc. Roy. Soc. Ser. B Biol. Sci. 154: 21-69, 1961. 132

Paton, W.D.M. and Rang, H.P.: The uptake of atropine and related drugs by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors. Proc. Roy. Soc. Ser. B Biol. Sci. 163: 1-44, 1965.

Paton, W.D.M.: Adrenergic receptors viewed in light of general receptor theories. Ann. N.Y. Acad. Sci. 139: 632-644, 1967.

Patterson, R.: The tracheal strip: Observations on the response of tracheal muscle. J. Allergy 2_9: 165- 172, 1958.

Pfeiffer, C.C.: Optical isomerism and pharmacological action, a generalization. Sci. 124: 29-31, 1956.

Powell, C.E. and Slater, I.H.: Blocking of inhibitory adrenergic receptors by a dichloro analog of isopro terenol. J. Pharmacol. Exp. Ther. 122: 480-488, 1958.

Rail, T.W. and West, T.C.: The potentiation of cardiac inotropic responses to norepinephrine by theophylline. J. Pharmacol. Exp. Ther. 139: 269-274, 1963.

Rang, H.P. and Ritter, J.M.: A new kind of drug anta gonism: Evidence that agonists cause a molecular change in acetylcholine receptors. Mol. Pharmacol. 5: 394-411, 1969.

Reiffenstein, R.J.: Effects of cocaine on the rate of contraction to noradrenaline in the cat spleen strip: mode of action of cocaine. Br. J. Pharmacol. Che mother. 32.: 591-597, 1968.

Robison, G.A., Butcher, R.W. and Sutherland, E.W.: On the relation of hormone receptors to adenyl cyclase. In Fundamental Concepts in Drug-Receptor Inter actions (J.F. Danielli, J.F.Moran and D.J. Triggle, • Eds.), 59-91, Academic Press, New York, 1970.

Schild, H.O.: Drug antagonism and pAx. Pharmacol Revs. 9: 242-246, 1957.

Scriabine, A., More, P.F., Iorio, L.C., Goldman, I.M., McShane, W.K. and Booher, K.D.: Quinterenol, anew beta adrenergic stimulant. J. Pharmacol. Exp. Ther. 162: 60-69, 1968. 133

Stein, W.D.: The movement of Molecules Across Cell Mem branes, Academic Press, New York, 1967.

Sutherland, E.W., Robison, G.A. and Butcher, R.W.: Some aspects of the biological role of adenosine 3',5'- monophosphate (cyclic AMP) . Circulation 37_: 2 79- 306, 1968.

Takagi, K. and Takayanagi, I.: Beta-adrenergic recep tors in various organs. Jap. J. Pharmacol. 20: 92-101, 1970.

Trendelenburg, U.: The effect of cocaine on the pace maker of isolated guinea-pig atria. J. Pharmacol. Exp. Ther. 161: 222-231, 1968.

Trendelenburg, U.: The pharmacological importance of the uptake mechanism for sympathomimetic amines. Prog. Brain Res. 31: 73-85, 1969.

VanDeripe, D.R., Ablad, B. and Moran, N.C.: 8-adrenergic blockade by four methyl substituted N-isopropyl- phenylethanolamines. Fed. Proc. 23_i 12 4, 19 64.

Van Rossum, J.M.: Cumulative dose-response curves. II. Techniques for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch. Int. Pharmacodyn. Ther. 143: 299-330, 1963.

Van Rossum, J.M. and Mujic, M.: Classification of sym pathomimetic drugs on the rabbit intestine. Arch. Int. Pharmacodyn. Ther. 155: 418-431, 1965.

Wenke, M., Lincova, D., Cepelik, J., Cernohorsky, M. and Hynie, S.: Some aspects of the action of beta adre nergic blocking drugs on adrenergic lipid mobiliza tion. Ann. N.Y. Acad. Sci. 139: 860-878, 1967.

Wilkenfeld, B.E. and Levy, B.: Adrenergic blocking pro perties of MJ 1999 and butoxamine on cardiac and vascular beta receptors. Arch. Int. Pharmacodyn. Ther. 176: 218-232, 1968.

Wilkenfeld, B.E. and Levy, B.: The effects of theophylline, diazoxide and imidazole on isoproterenol-induced inhibition of the rabbit ileum. J. Pharmacol. Exp. Ther. 169: 61-67, 1969. 134

Youmans, W.B. , Aumann, K.W. and Haney, Ii.F.: Relation of the various groups of the adrenalin molecule to its intestine inhibiting function in unanesthetized dogs. Am. J. Physiol. 126: 237-246, 1939.