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GARG, Bhagwan Dass, 1938- PHARMACOLOGICAL AND BIOCHEMICAL STUDIES ON DISPOSITION OF OPTICAL ISOMERS OF CATECHOLAMINES.

The Ohio State University, Ph.D., 1972 Pharmacology

University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED PHARMACOLOGICAL AND BIOCHEMICAL

STUDIES ON DISPOSITION OF OPTICAL

ISOMERS OF CATECHOLAMINES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Bhagwan Dass Garg, B.V.Sc.& A .H ., M.Sc. (V.M.&A.H.)

* * * * *

The Ohio State University 1972

Approved By

A dviser PLEASE NOTE:

Some pages may have

indistinct print.

Filmed as received.

University Microfilms, A Xerox Education Company ACKN OWLEDGM ENTS

I wish to express my sincere thanks and gratitude to Dr. Popat N.

Patil, my major adviser, for his valuable guidance and instructions throughout all phases of the work. For this I shall ever remain indebted to him .

My thanks are due to the members of my research advisory com­ mittee, Drs. H.H. Wolf, T. Sokolowski, and D.R. Feller, for their suggestions, constructive criticism, and critical appraisal of the work.

I am grateful to Dr. Robert D. Krell, a former colleague of mine, for his help and advice in many facets of this work.

My thanks are due to Haryana Agricultural University, Hissar,

India; Government of India, and U.S. Agency for International Develop­ ment for financial assistance.

I am particularly grateful to my wife, Swaraj, and my daughters,

Meena, Sadhna, and Meera, for their extreme patience, sacrifices, and encouragement during my four years separation from them. This dissertation is indeed appropriately dedicated to them.

ii VITA

February 20, 1938 Born - Sunam, Punjab, India

1959 B.V.Sc.&A.H. (Bachelor of Veterinary Medicine and Animal Husbandry), . Panjab University, Chandigarh

1959 - 1963 Lecturer, College of Veterinary Medi­ cine, Hissar, Plaryana, India

1963 - 1965 M .Sc. (Vet. Med. and A .H .), Punjab Agricultural University, Hissar, India

1965 - 1968 Assistant Professor, College of Veter­ inary Medicine, Hissar, Haryana, India

1968 - 1972 Doctoral Program, The Ohio State University, Columbus, Ohio

PUBLICATIONS

"Influence of Tropolone on the Magnitude and Duration of Action of Catecholamine Isomers", Pharmacologist, 12_: 306,1970.

"Phytochemical and Pharmacological Investigation of Anchusa Strigosa, an Indian Medicinal Plant", Ind.J.M ed.Res. 4: 185-192, 1970.

"Steric Aspects of Adrenergic Drugs .XVII. Influence of Tropolone on the Magnitude and Duration of Action of Catecholamine Isomers", Arch. int. pharmacodyn.189: 281-294, 1971.

iii FIELDS OF STUDY

Major Field: Pharmacology

Autonomic Nervous System

Structure - Activity Relationships of Adrenergic Drugs

Adrenergic Mechanisms TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... ii

VITA...... iii

LIST OF TABLES...... v iii

LIST OF ILLUSTRATIONS...... ix

Chapter I INTRODUCTION

Disposition of Catecholamines ...... 1 Enzymatic Inactivation, COMT ...... 5 Monoamine Oxidase ...... 9 Tissue Uptake of Catecholamines ...... 12 Extraneuronal Uptake (Uptake 2 )...... 16 Steric Aspects of Catecholamine Disposition ...... 17 Stereoselectivity of COMT ...... 19 S te re o sele c tiv ity of MAO ...... 20 Stereoselectivity of the Uptake Process for Optical Isomers of Epi and N E ...... 22 Statement of the Problem ...... 26

Chapter II METHODS AND MATERIALS

Rabbit Aorta, Oil Immersion Technique ...... 30 Experimental D esign...... 31 (a) Influence of COMT Inhibition on the Duration of Action of Catecholamine Isomers ...... 31 (b) Influence of Iproniazid on Magnitude and Duration of Action of Optical Isomers of NE ...... 33 (c) Influence of in vivo Inhibition of COMT on the Responses of Blood Pressure and Heart Rate of Rabbits to Optical Isomers of Epi ...... 34 Biochemical Studies ...... 36 Determination of the Accumulation of (-)- and (+)-NE- C by Heart Slices ...... ‘...... 36 In Vivo Infusion of (-)- and (+)-NE-^C ...... 38

v Determination of O-Methylated Products ...... 39 Schedule of Treatments in in vivo Experiments In M i c e ...... 39 Determination of Radiochemical Purity of NE-*-4C Isomers and (i)-Normetanephrine-^H ...... 41 Separation of M etabolites , Sample Preparation, and Liquid Scintillation Counting ...... 42 Preparation of the Ion-Exchange Columns ...... 44 Statistical A nalysis ...... 46 Drugs and Solutions ...... 46 C hapter III RESULTS Influence of Tropolone on the Magnitude and Duration of Action of Catecholamines on Rabbit Aortic Strips (Oil Immersion Technique) ...... 48 Influence of Tropolone on the in vivo Cardiovascular Responses to Epi Isom ers ...... 60 Influence of MAO Inhibition on the Magnitude and Duration of Action of NE Isomers on the Rabbit Aortic Strip (Oil Immersion Technique) ...... 63 In Vitro A ccum ulation of (-)- and (+)-NE-*-4C by Heart Slices ...... 65 Determination of Kinetic Constants of Accumulation of NE-*4C in H eart S l i c e s ...... 69 Effect of Guanethidine on Uptake of NE-^C Isomers in Heart Slices ...... 72 In vivo Accumulation and Retention of (-)- and (+)-NE-i4C in Mice H earts ...... 72 Disappearance of NE-*4C Isomers in Guanethidine or Reserpine Treated Anim als ...... 81 Disappearance of NE Isomers from Hearts of c^-MPT Treated M ice ...... 83 Disappearance of Isomers of NE-*4C from the Hearts of 6-Hydroxy Dopamine Treated Mice ...... 84 Determination of O-Methylated Metabolites of (-)- and (+)-NEJ4 C ...... 86

Chapter IV DISCUSSION

Kinetic Analysis of Accumulation of (-)- and (+)-NE-^4C by Heart Slices of Various Species of A nim als ...... 88 Retention of (-)- and (+)-NE-^4C by Mouse-Heart ...... 93 Norepinephrine Storage Pools ...... 96

vi Stereoselectivity of COMT ...... 99

C hapter V SUMMARY AND CONCLUSIONS...... 107

BIBLIOGRAPHY ...... Ill

v ii Filmed as received viii without page(s)

UNIVERSITY MICROFILMS. LIST OF ILLUSTRATIONS

Figure Page

1. A scheme showing the pathways of metabolism of norepinephrine and epinephrine ...... 4

2. Structural formulas for (-)- and (+)-isomers of norepinephrine and epinephrine ...... 18

3. Schematic illustration of time control experiments on rabbit aorta (oil-immersion technique) ...... 49

4. Influence of tropolone on the duration of action of norepinephrine isomers on rabbit aorta (oil-immersion te c h n iq u e )...... 51

5. Influence of tropolone on the duration of action of (+)-norepinephrine and dopamine on rabbit aorta (oil-immersion technique) ...... 55

6. Influence of tropolone on the duration of action of epinephrine isomers on rabbit aorta (oil-immersion te ch n iq u e)...... 59

7. Influence of U-0521 on the effect of (-)-epinephrine on the blood pressure and heart rate of rabbits ...... 61

8. Influence of U-0521 on the effect of (+)-epinephrine on the blood pressure and heart rate of rabbits ...... 62

9. Influence of iproniazid on the duration of action of norepinephrine isomers on rabbit aorta (oil-immersion 64 technique)......

10. Time-sequence analysis of the accumulation of (-)- and (+)-norepinephrine-*-^ by cat, rabbit and guinea pig heart slices...... 67

ix 11. A ccum ulation of (-)- and (+)-n o rep in ep h rin e- C by cat, rabbit and guinea pig heart slices at different concentrations of norepinephrine ...... 68

12. Kinetic analysis of the accumulation of (-)- and (+)-norepinephrine-^C by cat, rabbit, and guinea pig heart slices ...... 70

13. Accumulation of (-)- and (+)-norepinephrine-^C by normal and guanethidine treated cat, rabbit, and guinea pig heart slices ...... 73

14. Semi-logarithmic plot of the disappearance of (-)- and (+) -norepinephrine-*-^ C in vivo from the hearts of normal mice and mice treated with iproniazid andc<.-MPT ...... 74

15. Semi-logarithmic plot of the disappearance of (-)-/ (+)-/ and (t)-norepinephrine-^C in vivo from the h earts of ip ro n iazid treated m ic e ...... 78

16. Semi-logarithmic plot of the disappearance of (-)- and (+)-norepinephrine-^C in vivo from the hearts of mice treated with iproniazid, iproniazid and reserpine, and iproniazid and guanethidine ...... 82

x CHAPTER I

INTRODUCTION

Disposition of Catecholamines

Cannon and Rosenblueth (1933) described that sympatho-adrenal system acted as one functional unit and liberated one common hormone

"sympathin" . Sympathin then combined with either substance "E" or substance "I" in the effector cell to form hypothetical compounds,

"sympathin E" or "sympathin I" which had excitatory and inhibitory activities respectively. Bacq (1934) suggested that Cannon's "sympa­ thin E" was norepinephrine (NE) and that "sympathin I" was epineph­ rine (Epi). It was Euler (1946) who isolated NE from adrenergic nerves and provided a final proof that NE was the chemical mediator of the mammalian sympathetic nerves. The discovery of NE as the neuro­ transmitter together with the advances in biochemical and histochemi- cal techniques have caused a great upsurge of interest in the study of adrenergic mechanisms.

It has now been conclusively proven that NE is released from the sympathetic nerves as a neurotransmitter and acts locally while

Epi is discharged into the blood stream mainly from the adrenal medulla and also, to some extent, from the extra-adrenal'chromaffin tissue

1 and acts as a true hormone on distant organs. Naturally occuring material in splenic nerves was identified as (-)-NE by Euler (1948).

The uptake, binding and release of catecholamines has been extensive­ ly studied and reviewed (Axelrod, 1959, 1963, 1965, 1968, 1971; Zeller,

1959; Armstrong and McMillan, 1959; Molinoff and Axelrod, 1971;

Sandler and Ruthvin, 1968; Euler, 1956). In the present review, a brief introduction of the various routes of disposition of catecholamines and the steric aspects of drug action will be provided.

The disposition of the neuronally liberated transmitter differs from that of the circulating catecholamines mainly because of the differences in the manner of presentation of the compounds to the sites of inactivation rather than the differences in the mechanisms of inacti­ vation themselves. The various routes of disposition of catechol­ amines are:

1. Enzymatic inactivation by monoamine oxidase (MAO) or catechol-

O-methyl transferase (COMT) or both.

2. Extraneuronal binding to the neuroeffector or the surrounding connective tissue.

3. Uptake into the adrenergic nerves by the membrane pump.

4. Diffusion into the systemic circulation.

W hereas, uptake by the membrane pump and enzymatic oxida­ tion are more important mechanisms of disposition of neuronally released NE, inactivation by COMT and extraneuronal binding play more important roles for circulating catecholamines. The fate of neuronally released NE is different in tissues differing in the extent of sympathetic innervation. The synaptic gap in the neuroeffector sys­ tem of small blood vessels in the cat skeletal muscle has been reported to be 0.1 micron (Folkow et a l. ,1967). The similar gap in the rabbit pulmonary artery is 4.0 micron (Verity and Bevan, 1968; Su and Bevan, 1971). In the case of blood vessels of the skeletal muscle, therefore, the entire transmitter will be able to reach the receptor before being inactivated. In large elastic arteries, with a wide separation of neuroeffector junction, it is feasible that a portion of the transmitter is taken back into the adrenergic nerves before it reaches the receptor site. As compared to the small blood vessels, diffusion of the transmitter to the blood may be more important in the large arteries.

A general scheme for the major pathways of metabolism of Epi and NE is shown in Fig. 1. The different methods of disposition of these compounds are discussed individually in the following pages. 4

HO H HO H I I .C — C —NHCII. C— C-NHC1C. ^ C -C —N1ICH,

HO'HO HO­ OCH, OCH,

Epinephrine Conjugated motancphrine

HO :-c—oh C-OH

HOHO HO' OH OCH, OCH,

3 ,4-Dlhydroxymandcllc 4 -Hydroxy-3 - ni ethoxy- 4 - Hyd roxy - 3 - me thoxy - acid mandelic acid mandellc aldehyde

HO OH ■C—C— N il. ,C— C —N il,

no- HO" HO' OH OCH,

4-IIydroxy-3-methoxy- Norepinephrine Normetanophrine phcnylglycol

HO H COCH, III.’ H? V C-C—N—H C— C— NH, COOH H H II H HO RO OCH, OCH,

,W-Acetylnormetancphrine Conjugated normctanephrine Vanillic acid

Figure 1

Major pathways for metabolism of NE and Epi. 1. Catechol-O-methyl transferase. 2. Monoamine oxidase. Enzymatic Inactivation

COMT

An important original observation was recorded by Armstrong et al. (1957) who found in the human urine, 3-methoxy ,4-hydroxy mandelic acid (vanylmandelic acid, VMA) a major metabolite of Epi and NE. This finding pointed to O-methylation as a significant mech­ anism of inactivation of catecholamines. The problem was elegantly studied by Axelrod (1959,1963, and 1965). The properties of COMT were studied by Axelrod and Tomchick (1958). It require S-adenosyl methionine as the methyl donor and Mg for its action. Mg could* l~~h H-+ +*+■ ++ be replaced by other divalent ions such as Mn , CO , Zn , Fe , ++ ++ Cd , or Ni . All catecholamines examined could be O-methylated by the enzyme with almost the same velocity. Monophenols like phenylephrine did not serve as substrates. In vivo enzymatic-O- methylation was observed to occur only at the meta position. Creveling et al.. (1970) in vitro studied the properties of purified COMT prepara­ tion and found that O-methylation could occur both at para and meta positions depending upon the pH of the reaction mixture and the nature of the catechol substrate. The enzyme, COMT, is ubiquitously distributed in all mammalian species and also in some plants (Axelrod and Tomchick, 1958). It is present in highest concentration in liver and kidney. The enzyme occurs mainly in the soluble fraction of the cell but a small amount is also present in the microsomes (Inscoe etal./ 1965). Enzyme COMT is involved mainly in the metab­

olism of catecholamines outside the neuron because of its extraneu­

ronal distribution (Eisenfeld et al., 1967a,b) . However, some

amount of COMT has also been found to be present in the sympathetic

nerves of the cat nictitating membrane and the rat vas deferens (Jarrot,

1971b). On the basis of kinetic data of the neuronal and extraneuronal

COMT, the author has proposed that there may be two different en­

zymes with separate characteristics. S-adenosyl methionine is

normally present in tissues in suboptimal concentrations. Therefore,

the low activity of COMT under these conditions was generally missed

by earlier workers. The activity could be increased in the presence

of optimal concentrations of S-adenosyl methionine. Furthermore, an

appreciable reduction of COMT activity could be demonstrated on

denervation of sympathetically innervated organs. (Jarrot, 1971b).

A nderson and D 'lorio (1968) stu d ied th e electro p h o retic pattern of a

partially purified COMT from rat vas deferens. They observed that

there may be multiple forms of the enzyme. Axelrod and Vesell (1970)

reported the presence in the rat liver of two types of COMT which

differ on the basis of their electrophoretic mobility.

A xelrod, Senoh, and W itkop (1958) dem onstrated th a t in je c ted

Epi and NE were first O-methylated and later deaminated. That

O-methylation is the means of inactivation of these amines, is appar­

ent from the fact that norm eta nephrine formed by O-methylation of NE possesses 1/5 ^ of the activity of NE (Evarts et al. ,1958). The implica­ tion of this observation was that the effect of endogenous or exogenous

NE could be prolonged by inhibiting this enzyme. Polyphenols, e.g., pyrogallol and tropolone, are competitive inhibiters of COMT (Bacq

.et a l., 1959). W ylie e t a l. (1960) observed th a t adm in istratio n of pyrogallol in doses sufficient to inhibit COMT could significantly increase the duration of pressor effects and the electrical stimulation on the cat nictitating membrane.

Crout (1961), however, observed no potentiation of cardiovascular effects of NE in dogs after inhibition alone of COMT or both COMT and

MAO. Absence of potentiation does not prove, however, that a mech­ anism of inactivation is unimportant. Alternate pathways or diffusion of the agonist out of the biophase of the receptor may mask such potentiation (Kalsner and Nickerson, 1968 b).

Giles and Miller (1967a,b) reported that on rabbit atria U-0521

(3', 4'dihydroxy-C* -methyl propiophenone), a competitive inhibiter of

COMT, caused a parallel shift to the left of the dose response curve of Epi and isoproterenol. The effects of phenylephrine were not potentiated.

With the aid of oil immersion technique (Kalsner and Nickerson,

1968a), the disposition of NE and Epi has been studied in the rabbit aorta (Kalsner and Nickerson, 1969 a). The investigators observed that inhibition of COMT with tropolone significantly prolonged the relaxation in oil of the tissue contracted by Epi or NE.

O ssw ald et a l. (1970) stu d ied the fa te of Epi and NE in th e is o ­ lated venous strips of dogs using the oil immersion technique. They observed that O-methylation was relatively a minor route of disposition of these amines in this tissue while oxidative deamination represented the major inactivation pathway.

From the foregoing introduction, it appears that depending upon the tissue and the species, COMT could influence the fate of catechol­ amines . COMT could play an important role in large elastic arteries where the innervation is sparse and the synaptic gap is large. The role of COMT in relation to tissue morphology remains yet to be stu d ie d . Monoamine Oxidase

MAO is another enzyme which is involved in the metabolism of catecholamines. Besides catecholamines, the enzyme can deaminate a wide variety of other amines, e.g., tryptamine, tyramine, etc. The role of MAO in degradation of catecholamine has been studied by

Arm strong .et a l. (1957) and review ed by Axelrod (1959) and Kopin (1964).

MAO deaminates compounds in which the amine group is attached to the terminal carbon atom. N-methylation and ft -hydroxylation decrease the susceptibility of phenylethylamines to MAO (Blashko,

1952). Thus tyramine and dopamine are better substrates than Epi and

NE.

MAO is widely distributed in many animal species where it is present mainly in the outer membrane of the mitochondria (Schnaitman et al. ,1967) and in the NE storage vesicles of sympathetic neurons

(Roth and Stjarne, 1966 and de-Champlain, Mueller, and Axelrod, 1969).

Presence of MAO in sympathetic neurons has also been demonstrated by

Kopin and G orden (1963) and Smith (1966) on the b a s is of pharm acologi­ cal experiments and by Malmfors (1965) on histochemical investigations.

Recently, Jarrot and Iversen (1971) and Jarrot (1971a) demonstrated that MAO is present both extra- and intra-neuronally. Denervation of the'sympathetic nerves resulted only in partial fall of the enzymatic activity. It has been suggested by various workers that MAO from certain organs exists in multiple forms which differ in substrate specificities (Youdim et al. ,1969), inhibitor sensitivities (Johnston,

1968, and Hall .et al. ,1969) and kinetic properties (Jarrot, 1971a).

Many potent inhibitors of MAO have been introduced (Zeller, 1959;

Pletscher, 1966). Some compounds produce long-lasting, non-compet­ itive and irreversible inhibition of the enzyme, e.g., hydrazine derivatives including iproniazid (Zeller and Barsky, 1952). Other substances produce a short-lasting reversible inhibition, e.g., harmala alkaloids, harmine and harmaline. Many of these compounds are used in the therapy of psychic depression and in treatment of hyper­ tension. The mechanism of action of these drugs in the treatment of hypertension is not clear, since inhibition of MAO with iproniazid did not lead to potentiation or prolongation of the pharmacological effects of NE and other sympathomimetic amines (Griesmer, 1953).

There is a considerable evidence that MAO is involved in the regulation of intraneuronal level of NE (Kopin, 1964). Administration of iproniazid, a long-lasting MAO inhibiter, has been shown to result in increased levels of NE and other amines in brain (Spector jet al. ,1958;

Spector,1963) and certain peripheral tissues (Goldberg and Shideman,

1962, and Bhagat, 1963). It has been suggested that MAO serves to regulate the storage levels of NE in sympathetic nerve endings by destroying NE synthesized in excess of storage capacity (Brodie and

Beaven, 1963). There are, however, reports that inhibition of MAO did not result in a rise in the heart levels of NE in dogs or cats (Maling 11 et al. ,1962) or in mice (LeRoy and DeSchaepdryver, 1961).

To sum up the role of metabolic enzymes, it may be stated that neither MAO nor COMT is essential for termination of the physiological actions of catecholamine in vivo. COMT is mainly concerned with metabolism of extracellular catecholamines while MAO may serve to regulate the intraneuronal level of NE in sympathetic nerves. MAO is little concerned with the metabolism of extracellular catecholamines, except in the deamination of inactivated O-methylated amines formed from the administered catecholamines. The role of intraneuronal COMT

(Jarrot, 1971b) and extraneuronal MAO (Jarrot, 1971a) remains yet to be d e fin e d . 12

Tissue Uptake of Catecholamines

The ability of postganglionic sympathetic neurons to take up

neuronally released NE or exogenous catecholamines has long been

recognized. The subject has been extensively reviewed (Iversen, 1967,

1971; Axelrod, 1971). Under physiological conditions, the uptake

mechanism constitutes an important means of inactivation of the adren­

ergic transmitter. Axelrod, Weil-Malherbe and Tomchick (1959) dem- q onstrated that after small doses of IT-NE to mice, the unchanged

amine accumulated into the tissues and continued to disappear over a

long period of time. Whitby, Axelrod, and Weil-Malherbe (1961) re-investigated the problem by giving H-NE to cats and mice and

confirmed that tissue uptake operated to remove the circulating amine .

Using a fluorimeteric method of catecholamine assay, Stromblad and

Nickerson (1961) also demonstrated that tissue uptake was an impor­

tant mechanism for physiological disposition of catecholamines. NE

uptake in the isolated hearts or atria of the guinea pig, rabbit, dog, or rat has been reported by Muscholl (1960,1961), Burn and Burn (1961),

Axelrod et a l. (1962), Kopin et al. (1962), Iversen (1963) and Trendelen­

burg and Crout (1964). As a matter of fact, the process of uptake has

been demonstrated to occur in all peripheral tissues which are inner­

vated by adrenergic nerves. The uptake of catecholamines could not

be demonstrated in the brain tissue because of the presence of blood

brain b a rrie r. G low inski e t a l. (1965) and G low inski and Axelrod (1966) 13 circumvented this problem and demonstrated the uptake of NE in brain tissue after administration of catecholamines into the lateral v e n tric le .

Whitby et al. (1961) reported that a good correlation existed between the extent of uptake of ^H-NE and richness of sympathetic innervation of the tissues of the cat. That the uptake of NE was

solely restricted to adrenergic nerves is clear from experiments in which the denervated tissues fail to accumulate the exogenous NE.

(Hertting et al. ,1961; Hertting, 1965; Potter etal., 1965; Iversen,

1965; Iversen et al., 1966). W olfe M M * (1962) and W olfe and Potter

(1963) with the help of electron microscopic and radioautographic tech­ niques demonstrated that ^H-NE in the heart and pineal glands of the rat was localized in postganglionic sympathetic neurons. This obser­ vation was also supported by histochemical studies of Hamberger et al.

(1964); Malmfors (1965) and Hamberger and Matsuoka (1965). Gillespie and Kirpekar (1965a,b) combined the use of fluorescent histochemical technique and autoradiography and demonstrated that H-NE infused into the cat spleen was accumulated in the adrenergic nerve fibers which also contained endogenous NE. Further evidence that the uptake of catecholamines occurred in the adrenergic nerves came from the experiments of Hertting and Axelrod (1961) and Gillespie and Kirpekar O (1965 a,b) who demonstrated that H-NE was released from the cat spleen by stimulation of the splenic nerve. Various studies have demonstrated that uptake of catecholamines is an active process. Iversen (1963) showed that in the isolated rat heart, perfused with low concentration of NE (10-20 ng/ml), the concenr tration of NE in the heart was 30-40 times that in the perfusion medium.

If it is assumed that accumulation of catecholamines occurs only in the

sympathetic nerves in the hearts, and knowing that the nerves form a very small fraction of the heart weight, the actual ratio of concentra­ tion in the nerve to that in the medium must be very high, probably

10,000 to 1 or even more, (Iversen, 1963). Iversen also demonstrated that the process of uptake of catecholamines was a saturable one and obeyed the Michaelis-Menton equation for enzyme substrate interaction.

It is now believed that uptake of catecholamines occurs in two

steps; the first, influx of the amine across the neuronal cell membrane, and the second, incorporation of the amine into intracellular storage granules (Carlsson et al., 1963; Furchgott et al., 1963; and Stjarne,

1964). W akade and Furchgott (1968) stu d ied the m etabolic req u ire­ ments for the uptake and storage of NE by the isolated left atrium of the guinea pig. They reported that uptake was energy dependent. They also demonstrated that energy derived from either glycolysis of exo­ genous glucose or oxidation of noncarbohydrate endogenous substrates was adequate both for the inward transport of NE across the neuronal

cell membrane as well as for the subsequent incorporation into intra­ neuronal storage granules. The uptake of catecholamines is a relatively non-specific mech­ anism. Various amines structurally related to NE such as metaraminol are taken up by the very process which is responsible for transporting

NE. Metaraminol is not a substrate for either MAO or COMT and thus has provided a useful tool to study the properties of the NE uptake process. Tissues accumulate metaraminol by a process which is temperature, Na+ and K+ dependent and is inhibited by Ouabain

(Giachetti and Shore, 1966a, and Paton, 1971).

Carlsson et al. (1963) demonstrated that isolated storage particles from the adrenal medulla were able to accumulate Epi and NE from the surrounding medium. Euler and Lishajko (1963) demonstrated the uptake of NE by the bovine splenic nerve particles which were partially depleted of their endogenous NE content. Uptake by the iso­ lated nerve granules does not fully account for the uptake by the whole nerve and this is possibly the reason for hypothesizing a two stage mechanism of uptake by the neurone (Gillis, 1964). Extraneuronal Uptake (Uptake 2)

Iversen (1965) observed, during rat heart perfusion experiments,

that neuronal uptake of NE saturated at an external amine concentration

of 0 .2 yg/m l. When, however, the perfusion was continued at still

higher concentrations, an unexpectedly large uptake of NE was observed.

Iversen termed this second uptake as uptake 2 to differentiate it from

the first uptake which is known as uptakeL ightm an and Iversen (1969)

found that uptake£ also accounts for a substantial proportion of NE by

the perfused heart even at low concentrations, but any catecholamine

taken up by uptake 2 is rapidly metabolized. Thus the accumulation of

unchanged amine was observed only at high perfusion concentrations.

The two types of uptake differ in many respects. Uptake^ occurs only in the sympathetic nerves whereas uptake2 has been shown to occur in

cardiac muscle (Ehinger and Sporring, 1968; Farnebo and Malmfors,1969)

and smooth cells of spleen and blood vessels (Gillespie, Hamilton,

and Hosie, 1967, 1970; Avakian and Gillespie, 1968; Gillespie, 1968;

and Gillespie and Muir, 1970). Uptake 2 is easily washed out in the

tissues. It is inhibited by normetanephrine and metanephrine which

are very poor inhibiters of intraneuronal NE uptake and is not inhibited

by metaraminol and poorly so by cocaine (Iversen, 1965; Lightman and

Iversen, 1969). The affinity of (±)NE for uptake 2 was demonstrated to

1/374 that for uptakej. Uptake 2 favored the accumulation of Epi over that of NE. Uptake2 operates at all concentrations of catecholamines 17 to transport the amine into non-neuronal tissues in which it is sub­ sequently metabolized. This process probably serves to have physiol­ ogical importance in terminating the actions of NE after its release from the adrenergic nerve terminals and may have pharmacological signifi­ cance in limiting the actions of certain catecholamines (Vane, 1969;

Iversen, 1971).

Steric Aspects of Catecholamine Disposition

NE and Epi both possess one assymetric center at the beta carbon atom in the side chain, (Fig. 2). Thus, two optically active viz levo

(-) and dextro(+) rotatory forms exist for each of these drugs. Endo­ genously liberated amines, NE or Epi are levorotatory, (Euler,1948).

Adrenergic receptors can discriminate between the enantiomorphs of these and other sympathomimetic amines. As an example, (-)NE is almost 300 times more potent than (+)-NE on the rabbit aorta (Patil et a l. , 1971). It was found necessary to investigate whether the biochem­ ical mechanisms involved in the disposition of these amines are also able to discriminate between these isomers. Various investigators have attempted to find the answer to this question but the problem still remains to be solved. 18

H H -H U - o —C—-C— N. I I \H 'H OH H OH H

(-) -norepinephrine (-)-epinephrine

OH H OH H I r >H l / CHr HO C— C— N f HO' C — c — N' I I NN I I H H H HO H H HO

(+) -norepinephrine (+)-epinephrine

Figure 2

Structural formulas of (-)- and (+)-isomers of norepinephrine and epinephrine. 19

Stereoselectivity of COMT

Axelrod and Tomchick (1958) stu d ied the properties of a purified preparation of COMT obtained from rat liver. They found that (-)- and

(+)-isomers of Epi and (-)NE and dopamine were O-methylated at almost an equal rate thus indicating that the enzyme COMT lacks stereoselec­ tivity. Meshi et al.(1970) studied the metabolism of trimetoquinol

[JL“1“ (3 , 4 ,5-trimethoxybenzyl ) - 6 , 7-dihydroxy-l ,2 ,3 ,4-tetrahydroiso- quinoline], a potent bronchodilator and its (+)-antimer which is void of any pharmacological activity. They found that in rat and guinea pig, the maximum rate of O-methylation was significantly greater for the (-)- isomer than for the (+)-isomer. Since trimetoquinol is also a catechol, the O-methylation was considered to be carried out by COMT which was therefore found to be stereoselective in this case. Smissman and Bor- chardt (1971 a,b) synthesized conformationally rigid decalin and decalol analogues of pi -methyl dopamine and tested for their relative O-methyla­ tion by COMT. They concluded that fc-hydroxyl group played an impor­ tant role in determining the preferential conformation of the substrate for

COMT activity. Iversen and co-workers (1971) recently studied the rela- tive in vivo O-methylation of (-)-and (+)-NE-^C in mouse andrat. They found a significantly higher proportion of (+)-NEthan (-)-NE was metabo­ lized to normetanephrine in the whole mouse, rat heart, and rat brain. This observation implies a reverse stereoselectivity for COMT. Thus, it is clear that the few studies which have been conducted to determine the 20 stereospecificity of COMT are all at variance with each other.

Stereoselectivity of Monoamine Oxidase

MAO is intimately involved in the regulation of the neurotrans­ mitter at the neuro-effector sites. It is now increasingly realized that there are multiple forms of MAO. It was demonstrated by Blashko et al.

(1937 a) that MAO obtained from guinea pig liver oxidized (-)Epi to a greater extent than (+)-Epi. Pratesi and Blashko (1959) reported that the two stereo-isomers of ^-hydroxyphenethylamine were oxidized at almost an equal rate by rabbit liver. Guinea pig liver extracts on the other hand oxidized (+)-form more rapidly than the (-)-form. They also observed the (+)- and (-)- isomers of amphetamine were almost equi- active as inhibiters of rabbit liver MAO whereas (+)-amphetamine was more potent inhibiter of guinea pig liver MAO. Kynuramine oxidation by rat liver MAO was inhibited more by the (+)-isomers of amphetamine,

2 ,4-dichloroamphetamine, 4-chloroamphetamine and 3,4-dichloro- amphetamine as compared to by their corresponding (-)-isomers (Fuller and W alters, 1965). Substitution of both hydrogen atoms at thecrt- carbon of the NE molecule by deuterium did not result in any alteration of its effect on cat nictitating membrane while a similar substitution in the tyramine molecule resulted in a marked potentiation (Belleau and

Burba, 1961). The authors interpreted that the enhancement of the tyramine effect was due to the fact that deuteriated tyramine was a

poorer substrate than tyramine for MAO. Giachetti and Shore (1966 b) made an elaborate study on the stereoselectivity of MAO using several

substrates. It was observed that (-)-isomers of Epi, NE, and octopa- mine are better substrates for MAO obtained from rat liver and brain and rabbit heart than the corresponding (+)-isomers. Another sugges­ tion that the MAO is stereoselective has been put forth by Knoll et a l.

(1968) as they showed that (-)-isomer of the compound, phenyl isopropyl methyl propylamine (E 250) is considerably more potent than the corres­

ponding (+)-isomer as inhibiter of the enzyme.

From the foregoing introduction, it appears that MAO or COMT manifest variations between tissues of the same species and between different species with regards to their stereoselectivity. Therefore in any study with catecholamines these factors should be borne in mind. 22

Stereoselectivity of the Uptake Process for Optical Isomers of Epi and NE

The stereochemical specificity of the uptake of NE into the adrenergic nerves has remained a controversial subject. Kopin and

Bridgers (1963) studied the accumulation and disappearance of the iso­ mers of NE after simultaneous administration of an equal mixture of

(t)-NE-3H and (-)-NE-4C or (t)-NE-3H and (-)-NE-4Cto rats. They 3 14 determined the ratio of H / C in heart and spleen 1 or 24 hours later.

They found that one hour after administration of labeled drugs, both isomers are taken up and bound to an equal extent while after 24 hours,

(-)-isomer was retained to a greater extent that (+)-antimer. It was con­ cluded that neuronal membrane is not stereoselective while the storage p ro cess i s . S ubsequently M aickel e t a l. (1963) and Beaven an d M aick el

(1964) reported that 5 minutes after i.v. injection of (1) NE-^H, (-)-iso- mer was taken up 11 times more than the (+)-isomer in the rat heart.

A study on the kinetic analysis of the uptake of NE isomers in the isolated perfused rat heart was made by Iversen (1963). The

Michaelis constant, km for (-)-NE was 1/5^ that for (+)-NE indicating five fold stereoselectivity of the membrane pump for (-)-NE. The V max for (+)-NE was twice that of (-)-NE. A study on the uptake of Epi isomers on the rat heart revealed three fold selectivity for the (-)- isomer (Iversen, 1965).

Crout (1964) stu d ied the accum ulation of H-NE by th e guinea pig heart after an intravenous injection of the appropriate non-labelled 23 isomer. He observed that both isomers are taken up and retained for about 1 hour to an equal degree.

AndSn (1964) reported a three fold s te re o se le c tiv e uptake of Epi isomers favoring the (-)-form in the mouse heart. Westphal (1965) analyzed the accumulation of Epi isomers in rat heart and liver. In both the tissues, a stereoselectivity for (-)-isomer by the storage process was reported. Mueller and Schideman (1964) investigated the uptake of NE isomers in cat atria and reported that the uptake mech­ anism in this tissue was not stereoselective. The same authors (in

1967) reinvestigated the problem using lower concentrations of these isomers and showed that at these concentrations, (-)-NE was taken up significantly more than (+)-NE.

The stereoselectivity of the uptake process continues to be investigated in a number of laboratories. Many different preparations and methods have been employed. Prenylamine, an agent which de­ pletes the endogenous amines from the adrenergic nerves, but does not damage the amine storing vesicles when administered to rabbits, caused greater accumulation and retention of the (-)-isomers of NE and

Epi over the corresponding (+)-isomers (Mackenna, 1965).

Draskoczy and Trendelenburg (1968) reported that in isolated perfused rabbit heart, the arteriovenous difference for (-)-NE was twice that of (+)-NE when the isomers were perfused at a concentration of

200 ng/ml. This selectivity was abolished in hearts of reserpine- 24 treated animals (Trendelenburg and Draskoczy, 1970) indicating that the observed stereoselectivity was a function of vesicles rather than the neuronal membrane.

Euler and Lishajko (1965) pretreated the rabbits with decaborane, an inhibiter of dopamine-£-hydroxylase in order to deplete the tissues of endogenous NE. Isomers of Epi were then simultaneously injected intramuscularly with (-)-NE to cause an equal vasoconstriction. They found that more (-) than (+) Epi accumulated both in the whole heart and in microsomal fraction indicating stereoselectivity favoring the (-)- isomer both at the neuronal and at the vesicular level.

Hendley and Snyder (1971) studied the selectivity of uptake of NE isomers in various tissues of the rabbit. Selectivity for (-)-NE was reported in vas deferens and iris/ciliary body, while a stereoselective reversal was found in the aorta, vena cavae, and the heart.

Sachs (1970) reported lack of stereoselectivity in the mouse heart.

Iversen and colleagues (1971) reported that rate of uptake of (-)-NE was significantly more than that of (+)-NE in isolated perfused hearts of reserpinized mice and rats when labelled amines were perfused but no selectivity was observed in guinea pig hearts. They also reported that uptake of NE was stereoselective in the hypothalamus but not in the corpus striatum of rat brains.

Considerable body of evidence suggests that binding of catechol­ amines in the storage granules is stereoselective. Carlsson et a l.(1963) demonstrated that isolated vesicles from the adrenal medulla took up twice as much (-)-Epi as (+)-Epi. Isolated bovine splenic nerve granules could selectively take up(-)-NE 5-7 times more than (+)- form

(Euler and Lishajko, 1964; Stjarne and Euler, 1965; Euler and Lishajko,

1967). Potter and Axelrod (1963), however, were unable to demonstrate any stereoselective preference for the NE isomers of the isolated nerve vesicles from the rat heart. Thus it appears that stereoselectivity of uptake is not a common phenomenon. It differs from tissue to tissue within the same species and also from species to species.

Stereochemical specificity of uptakeg for Epi and NE isomers was studied by Iversen (1965) in isolated perfused rat heart. Hearts were perfused for two minutes with (-)-NE, (+)-NE or (±)—NE at a concentration of 5 pg/ml. The uptake of the racemic form and that of the two enantiomers of NE was found to be equal. Similar experiment on the enantiomers of Epi did not reveal any significant difference between uptakes of (-) or (±)-Epi. It was therefore concluded that this extraneuronal uptake of catecholamines lacked stereoselectivity. No other investigations on the stereoselectivity of extraneuronal uptake appear to have been undertaken. Statement of the Problem

It is clear from the foregoing introduction that there are numerous controversies and inconsistencies in the existing literature as to whether the routes of disposition of catecholamines are stereoselective

or not. Very few studies have been carried out to test the stereoselec­

tivity of COMT and whatever information exists is controversial. It was therefore desirable to determine the stereoselectivity, if any, of this

enzyme. It was decided to examine the problem in two different ways:

(1) Pharmacologically: utilizing the oil immersion technique of Kalsner and Nickerson (1968a) and studying the rate of relaxation in oil of rabbit aortic strip contracted by (-) or (+) isomers of catecholamines before and after inhibition of COMT with tropolone. (2) Biochemically: in vivo

determining the O-methylated products formed from injected (-)-or(+)-

NE-^C. Both these techniques are simple and straight forward.

Another aim of this study was to ascertain whether or not inhibi­

tion of COMT leads to potentiation or prolongation of the pressor

effects of equiactive doses of Epi isomers in vivo. This would also

provide another means to study the stereochemical specificity of

COMT. A new COMT inhibiter, 3' ,4'-dihydroxy-2-methyl propiophen-

one (U-0521) (Giles and Miller, 1967a,b,c) will be used for in vivo

inhibition of COMT. Rabbit heart rate and blood pressure will be used

as parameters for studying the in vivo effects of Epi isomers. 27

Steric aspects of mechanism of uptake of catecholamines are

controversial. While it may be true that intraneuronal uptake of

catecholamine is stereospecific in rats and mice, but not in guinea pigs,

(Iversen et a l., 1971), this seems difficult to reconcile with intuitive logic. Moreover, there is no convincing explanation put forth by any worker for these discrepancies. Many workers (Iversen, 1963, 1965;

Snyder et al., 1968) have made use of the kinetic parameters Km for the affinity and V max for the maximum rate of uptake of isomers of catechol­ amines . In most instances, if the affinity (1/Km) is low for one isomer,

Vmex has been found to be higher or the vice-versa. Under these

circumstances, dependency on either one of these parameters would lead to incorrect interpretation of the data. To further explain this

point, the original Michaelis-Menton equation is reproduced below:

v V max FS1 (i) [S] + Km w here v = initial rate of reaction (here, rate of uptake), and

[S] = substrate concentration.

At very low substrate concentrations, i.e ., when [S] (<,Km, the equation (i) can be simplified to:

v = V max [S] (ii) Km 28

Since V max is constant, v^[S]. Km

Therefore, at very low substrate concentrations, the initial velocity is directly proportional to substrate concentrations.

At very high substrate concentrations, when [S]>/Km, the

equation (i) may be simplified to:

v = V max [S] = V max [S] (iii)

At moderate concentrations such as used in this study, both Km and Vmax could contribute to the net uptake. Under these conditions, ratio V max/Km, rather than either V max or Km alone is a better index of the initial rate of accumulation of NE. Keeping this in view, it was intended to investigate the accumulation of recently made available

labelled isomers of NE in the heart slices of different species.

Rabbits, cats and guinea pigs will be selected for the present study.

Kinetic parameters, Km, Vmax, and Vmax/Km will be determined. The differences between the uptake of isomers of NE in any given species and the differences between the species will be re-examined. An attempt will also be made to dissociate the study of uptake of NE by the neuronal membrane from that by the storage vesicles, inhibiting the latter with guanethidine.

In most of the previous studies designed to investigate the up­ take, storage or release of NE, use has been made of racemic NE-^H as a tracer of endogenous NE. While the endogenous transmitter is a pure (-)-isomer of NE, the labelled tracer used for these studies was a mixture of (-)- and (+)- NE. It is being increasingly realized that both (-) and (+)-isomers may not undergo the same fate in the body. . O The presence of (+)-isomer in the (-) -H-NE might therefore have exerted a distorting influence. Previous work may have to be re-eval­ uated in this light. It was therefore an aim of this study to re-inves- tigate certain features of uptake of NE keeping into consideration and circumventing where possible the problems faced by earlier investigators.

In a further attempt to study the storage and binding of NE, the rate of disappearance of (-)-^C-NE and (+)-^C-NE after in vivo infusion will be studied in mouse hearts. Effect of various drugs which are known to alter the uptake, storage and release of catechol­ amines in the adrenergic nerves will be studied in order to expose any selectivity of accumulation or storage. CHAPTER II

METHODS AND MATERIALS

Rabbit Aorta, Oil Immersion Technique

The technique described by Kalsner and Nickerson (1968 a) was used. Female albino rabbits (Kings Whale Rabbitry, Mt. Vernon, Ohio) weighing 3-4 lbs. were used in this series of experiments. The animals were killed by air embolism introduced through one of the marginal ear veins. The thoracic aorta was quickly and gently excised and the adherent fat and loose connective tissue removed. Spiral strips were prepared according to the method of Furchgott and Bhadra- kom (1953). Two strips, 25 mm x 2 mm were cut and suspended simul­ taneously under 1.5 gm tension in two separate 10 ml jacketed tissue baths containing physiological salt solution (PSS) of the following composition at 37.5-38° C.: NaCl 118 mM, KC1 4.7 mM, CaCl 2 •

2 H20 1.9 mM, NaHCOg 25 mM, MgCl 2 . 6 H20 0.5 mM, NaH^Po^

H20 1 mM and glucose 11 mM. Disodiumethylenediamenetetraacetic acid (EDTA), 10 pg/litre was added to retard the spontaneous oxidation of catecholamines. The chemicals were dissolved in double distilled demineralized water. The baths and the stock salt solution were kept aerated with a mixture of 95% 0 2 and 5% C02< Throughout the

30 procedure, from isolating the aorta till mounting the strips in the bath, the tissues were kept moist with PSS. The drug-induced responses were recorded via isotonic myograph on a physiograph (E&M Instru­ ments C o.).

Experimental Design

(a) Influence of COMT inhibition on the duration of action of catechol­ amine isomers.

Aortic strips were allowed to equilibrate for 90 minutes. Since the influence of enzyme COMT was to be studied, all other known routes of drug disposition were blocked, (i) MAO was blocked by irreversible inhibiter, iproniazid, (10~^M), (Kalsner and Nickerson,

1968a). At the end of the equilibration period, strips were exposed to the inhibiter for 30 minutes and washed several times, (ii) Thirty minutes after the last wash, neuronal uptake was blocked by cocaine,

(10“®M), (Garg et a l., 1971). (iii) Beta adrenergic receptors were blocked by propranolol (10- 6 M), (Garg et a l., 1971). The tissue contact time for cocaine and propranolol was 15 and 10 minutes respectively.

Cocaine was added 5 minutes prior to propranolol, (iv) On one strip a

(-)-isomer was tested while the other was used for the (+)-isomer when comparison was to be made between (-)- and (+)-isomers. When (+)- isomer and corresponding desoxy derivatives were compared, one strip 32 was used for (+)-isomer and the other for desoxy compound. It was

“ 8 c found that 10 M (-)-NE and 2 x 10- M (+)-NE were equiactive and

produced approximately 50% contraction of their maxima, (Patil et a l.,

1971). Dopamine and (+)-NE were found to be equipotent (Patil, 1971).

Similar equiactive concentrations for Epi isomers were found to be

10- ^M for (-)-Epi, 3 x 10~7M for (+)~Epi (Garg et a l., 1971). Equi­

active concentrations ofj^-methyl-NE (cobefrin) isomers were deter­

mined in separate series of experiments. It was found that cobefrin in _7 a concentration of 10 M produced contraction of the rabbit aortic strip which was 50% of its maxima. (+)-Cobefrin was about l/lOOO 1-*1 le s s

active in contracting this tissue. In this pair of drugs, therefore, a —7 comparison was made between 10 M (-)-cobefrin and a 1:4 mixture of

(-)- and (+)-cobefrin, (4 x 10” 7 M). (v) Ten minutes after the contrac­

tion produced by the agonists reached a plateau, the tissue-bath was

drained and filled quickly with the previously oxygenated warm (37°C)

mineral oil and the relaxation of the tissue was observed. When tis­

sue relaxed more than 50 percent of the original height, the oil was

drained and tissue was washed several times with the PSS. (vi) Fif­

teen minutes after the last wash, tissue was exposed to tropolone

(10“^M), cocaine (10~^M) and propranolol (10"®M). The total tissue

contact time for tropolone, cocaine and propranolol was 20, 15, and

10 minutes respectively. Cocaine was added five m os prior to

propranolol but five minutes after tropolone. (vii) Ti..: same 33 concentration of the agonist as used before tropolone was repeated,

(viii) Again, when contraction reached the plateau, the tissue bath was drained and filled with liquid mineral oil and relaxation was re c o rd ed .

The height of the contraction and the duration for 50 percent relaxation were recorded. The experiments were repeated several times so that the average values and standard errors of the mean (SEM) could be calculated.

(b) Influence of iproniazid on magnitude and duration of action of optical isomers of NE.

Rabbit aortic strips were prepared, mounted in the tissue bath and allowed to equilibrate as described above. The design of experi­ ment was modified as described below.

(i) After usual equilibration, the tissues were exposed to tropolone

(10~^M), cocaine (10- ^M) and propranolol (10“®M). The total tissue contact time was 20 minutes, 15 minutes, and 10 minutes for tropolone, cocaine and propranolol respectively.

(ii) Ten minutes after propranolol, one strip was exposed to (-)-NE

(10 ®M) while the other tissue was contracted with (+)-NE (2 x 10 ®M).

(iii) Ten-minutes after the contraction reached a plateau, the baths were drained and quickly filled with liquid mineral oil and the relaxa­ tion recorded.

(iv) After the tissues had relaxed more than 50 percent of the original height, the baths were drained and washed several times with the PSS.

(v) Ten minutes after the last wash, the tissues were exposed to -4 iproniazid (10 M). Thirty minutes were allowed for iproniazid tissue c o n ta c t.

(vi) Baths were again drained and washed several times during the next

30 minute period.

(vii) Ten minutes after the last wash, the tissues were again exposed to tropolone (10 ^M), cocaine (10 ^M), and propranolol for 20, 15, and

1 0 minutes respectively as was done before iproniazid treatment.

(viii) Ten minutes after propranolol, the tissues were again contracted with isomers in concentrations identical to those used before iproniazid treatm en t.

(ix) Ten minutes after the contractions reached a plateau, the baths were drained and quickly filled with liquid mineral oil as described before and duration for 50 percent relaxation was recorded.

Net increase in height and duration for 50 percent relaxation due to iproniazid treatment was calculated for both isomers and the means compared by student's t-test.

(c) Influence of in vivo inhibition of COMT on the responses of blood pressure and heart rate of rabbits to optical isomers of Epi.

Female albino rabbits weighing 1.5-2.5 kg were used. Animals were anaesthetized with Dial with urethane (Ciba, Summit, N .J.), 0.6 ml/kg administered intraperitoneally. Animals were pretreated with 35 reserpine 3 mg/kg i.p . 16-24 hours before the experiment. Vagi were sectioned. Carotid blood pressure and heart rate were recorded on a physiograph through a pressure transducer. Marginal ear vein was cannulated for drug injections.

Experimental Design:

After the preparation was allowed to stabilize for 30 minutes, cocaine, 10 mg/kg was slowly infused over a period of 5 minutes. Five minutes after the infusion of cocaine, either (-)-Epi, 1 pg/kg or (+)-Epi

1 0 0 p g /k g in 1 ml volume, followed by 1 ml of normal saline was injected i.v . and the effect on heart rate and blood pressure recorded. Ten minutes after the blood pressure and heart rate returned to normal,

U-0521 was injected in a dose of either 50 mg/kg or 100 mg/kg. Five minutes after U-0521 injection, the same dose of the isomer of the Epi as injected before U-0521 treatment was repeated. The differences in the magnitude of duration of cardiovascular effects were recorded. The experiments were repeated a sufficient number of times to calculate the means and standard error. 36

Biochemical Studies

Determination of the Accumulation of (-)- and (+)-NE-^Cby Heart Slices

Female albino rabbits, 3-4 lb s., (Kings Whale Rabbitry, Mt.

Vernon, Ohio), female albino guinea pigs, 250-300 g (Glenn Carr

Farms, Columbus, Ohio) and cats of either sex, 2.5-4 kg (Hubert

Becker, Celina, Ohio), were used in this part of the study. Rabbits and guinea pigs were sacrificed by a blow on the head while cats were killed by an overdose of pentobarbital sodium (50 mg/kg). The hearts were quickly removed. The atria were discarded and the ventricular portion was cut in two approximately equal halves . The ventricular slices were prepared with a tissue microtome (Stadie and Riggs, 1944).

Slices of the pericardium were removed. All the heart slices required for each experiment (six or eight slices) were obtained from one rabbit or cat heart. Guinea pig heart being small, sometimes yielded only four slices, so the required number of slices was obtained from two animals. The slices were paired between (-)- and (+)-isomers of NE-\l.

All data was obtained from the paired slices. Throughout the procedure the tissues were kept moist with PSS at room temperature.

After rinsing the slices a few times in the PSS, the slices were immersed in 20 ml beakers containing 5 ml of PSS and shaken 90 oscil­ lations per min in a Dubnoff metabolic incubator at 37.5°C under an atmos­ phere of 95% O2 a n d 5 %CC>2 . The beakers also contained tropolone (10~\l) and iproniazid (10- ^M) to inhibit COMT and MAO respectively. After 30 minutes preincubation and tissue contact with the inhibiters, vary­ ing concentrations of either (-)- and (+)-NE-^C were added to the beakers and incubation continued for 5 to 40 minutes. Following incubation, the slices were rinsed 3 times, with ice-cold PSS, blotted on filter paper, weighed and transferred to 2 0 ml beakers which con­

tained 5 ml of ice-cold O. 4 NHCIO 4 . The slices were homogenized with an Ultra-Turrax (Brinkman Instruments, Westbury, New York) for

20 sec at moderate speed. The Ultra-Turrax was rinsed with 2 ml of

0.4 NHCIO^ and this washing was added to the homogenate. The homogenates were centrifuged for 1 0 min at moderate speed in a clinical

centrifuge at room temperature. The resulting supernatant was filtered under suction and the pellet resuspended in 0.5 ml of 0.4 NHCIO 4

and recentrifuged for another 10 min. The second supernatant was mixed with the first and the final volume was made up to 8 ml. Since both MAO and COMT were inhibited, no metabolites were expected.

It was therefore assumed that all radioactivity was due to unchanged

NE-^C. An aliquot of 2 ml of the filtered homogenate was counted in

13 ml of Aquasol (New England Nuclear, Boston, M assachusetts) in a

Packard-Tricarb Liquid Scintillation Spectrometer (Model 3375) for two

20 min periods. Instrument efficiency, as determined by internal

standardization for was 76 percent and the results were corrected accordingly. Tissue:medium ratios for NE-^C were calculated as counts per minute (CPM) per g wet weight heart/CPM per ml of the 38 m edium .

In some experiments, the animals were pretreated with guanethi- dine 20 mg/kg, 16-24 hours prior to experiment. The slices were pre­ pared and processed in a manner identical to that described for control a n im a ls .

In vivo infusion of (-)- and (+)-NE-^C

Male albino ICR mice (Harlan Industries, Cumberland, Indiana) weighing 16-26 g were used in this study. Each animal was infused with either (-)- or (+)- NE-^C, via one of the lateral tail veins. The concentration of each isomer infused was 0 . 8 mg/kg which corre­ sponded to 0 .ljiCi /0 .lml/20g body wt. Infusion was constant at a rate of 0.2ml/min (Harvard Infusion Pump, Harvard Apparatus Co., Inc.,

M illis, M ass., Model 901) and required 25-40 sec for completion.

Mice were decapitated at varying periods post infusion (5 min to

24 hours). The hearts were quickly removed, cleaned of extraneous tissue, minced with scissors and rinsed 5 times in ice-cold 0.9% sodium chloride, blotted on filtered paper, weighed and transferred to 20 ml beakers containing 5 ml of 5% ice-cold trichloroacetic acid and

0.2 ml of 10% EDTA. Tissues were homogenized with Ultra-Turrax and the homogenate centrifuged and filtered as described for slices.

Separation of the m etabolites, sample preparation and liquid scintillation 39

counting was carried out as described later in this chapter.

Determination of O-methylated products

The O-methylated products of (-)- and (+)-NE-^C were deter­

mined in order to assay the activity of the enzyme COMT and thus to

study the stereochemical selectivity. Mice were pretreated with

iproniazid, 1 0 0 mg/kg about 12-16 hours before the experiment in order

to inhibit MAO. The mice received (-)- or (+)- NE-^C, 40 yig/kg in a

constant intravenous infusion. Ten minutes post-infusion, the animals were decapitated and their hearts removed. Hearts from five different animals were pooled together and this constituted one sample. Hearts were then rinsed, blotted dry, weighed, homogenized and filtered as described above. Separation of O-methylated derivatives was accom­ plished as described below.

Schedule of treatments in in vivo experiments in mice

1. MAO inhibition was accomplished by administering iproniazid

100 mg/kg, i.p ., 16-20 hours prior to infusion of NE-^C isomers.

Effect of all other pharmacological treatments was studied in MAO inhibited animals.

2. Guanethidine 20 m g/kg,i.p., was given 8-10 hours before iproniazid. NE-^C isomers were infused 24-30 hours after guanethi­ d in e . 40

3. Reserpine 5 mg/kg i.p. was given 8-10 hours before iproniazid in order to inhibit vesicular uptake and to observe whether

or not guanethidine acted in a manner similar to reserpine or not.

NE-^C isomers were infused 24-30 hours after reserpine and animals were sacrificed 15 minutes-4 hours thereafter.

4. Tyrosine-hydroxylase was inhibited by the administration of

two doses of d-methyl-p-tyrosine (g^MPT), 200 mg/kg each, 3 hours 14 apart. The last dose of o(MPT was given 1 hour before NE- C infusion.

Iproniazid was given 16-20 hours before the experiment.

5. Chemical sympathectomy of the heart was accomplished by

the administration of 2 doses of 6 -hydroxy dopamine, 75 mg/kg each i.p . at an interval of 1 hour, the last dose given about 24 hours before

NE-^C infusion. Iproniazid was given 16-20 hours before the

experiment.

6. In some experiments, (-)-NE-^C and (+)-NE-^C were mixed in equal amounts and (i)-NE-4C thus prepared was infused. Iproniazid was given 16-20 hours before the infusion of (i)-NE-^C. 41

Determination of radiochemical purity of NE-^C isomers and (±)-normetanephrine-3 H

Thin layer chromatography (TLC) was used to ascertain the radio­ chemical purity of the NE-^C isomers and (±)-^H-normetanephrine.

Five x 20 cm plates of silica gel were used. The solvent system used for TLC was butanolracetic acid:water (12:5:3). A spc ,1 of concentrated solution of cold (-)- or (+)-NE or (t)-normetanephrine, approximately

10-20 pg was used as a carrier. Approximately 1 jiCi of either NE-^C O isomer or normetanephrine- H was spotted on the top of corresponding carrier. The plates were air dried and developed in iodine chamber.

Rj values of (-)- or (+)-NE-^C or norm etanephrine-w ere found to be identical to those of the standards. In some cases, the silica gel from the plates was scraped off in 1 cm blocks and the scrapings were placed in separate counting vials with 2 ml of methanol and 13 ml of

Aquasol. The samples were counted for two 15 minutes periods in a

Packard Tricarb Liquid Scintillation Spectrometer. Four different batches of NE-.^C isomers were tested. No significant amounts of radiochemi­ cal decomposition products could be detected in any batch of NE-^C isomers. There were, however, some impurities (less than 10%) in the o 3 normetanephrine-°H sample. Purification of normetanephrine- H was accomplished by ion-exchange chromatography. Decomposition products did not bind to the columns and were eluted much earlier where-

3 as normetanephrine- H was eluted with 3 NHC1. Pattern of elution of 42 O normetanephrine- H was studied. Since a comparison was to be made between O-methylated products formed from (-)-NE^^C and (+)-NE-^C, absolute values of the metabolites formed were not critical. Moreover,

3 normetanephrine- H was used only as a standard for studying the pat­ tern of release of normetanephrine from the ion-exchange columns.

Therefore, no attempt was made to find out the exact purity of normetanephrine-^H.

Separation of metabolites, sample preparation and liquid scintillation counting

The filtered trichloroacetic acid homogenates of hearts were placed in 20 ml beakers containing 10 mg EDTA. The pH of the acid extracts was adjusted between 6 .2-6.5 with 2 NNaOH. The unchanged

NE-^C was separated from contaminating metabolites by passing the slightly acidic extracts through strong ion-exchange columns prepared from Amberlite CG-120, Na+ form according to the method of Haggendal

(1962a,b) slightly modified.

The preparation of the resin and the columns with the modifica­ tions made is described below.

1. Amberlite CG-120, 200-400 mesh, Na+ form, (Mallinckrodt chemical works) was poured in 500 ml beakers. This is a strongly acidic, sulfonated polystyrene type, cation exchange resin.

2. The resin was mixed with 2 volumes of double distilled 43 demineralized water, distilled again against EDTA and was allowed to stand until all the resin except the "fines" settle to the bottom of the beaker. The cloudy supernatant was decanted off. This procedure was repeated until the supernatant was clear. This usually required 20-25 w a sh in g s .

3. The resin was allowed to stand in water overnight to allow for its complete expansion.

4. The resin was washed twice with two volumes of 2 N NaOH containing 1% EDTA.

5. The'"NaOH was removed from the resin by washing with water.

The washing was continued until the supernatant was almost neutral

(checked with pH paper).

6 . The resin was then washed thrice with two volumes of 3 NHC1.

7. The HC1 was removed from the resin by washing with water until the supernatant was neutral (again checked with pH paper).

8 . The resin thus prepared was stored in water until needed to prepare columns. 44

Preparation of the ion-exchange columns

Glass columns, 9 mm dia x 150 cm height (Fisher and Porter C o.,

Warminster, Pa.) to which a 60 ml glass reservoir was attached were

u se d .

Step 1. The resin was added to the columns in a slurry to a

height of 35 mm (at neutral pH).

Step 2. Twenty ml of 2 N NaOH containing 1% EDTA was passed

through the columns.

Step 3. Columns were washed of the residual NaOH with

purified water containing 0.1% EDTA (about 15 ml of water was general­ ly u s e d ).

Step 4. Twenty ml of 2 NHC1 was passed through the columns.

Step 5. HC1 was washed off the columns with purified water

(about 2 0 m l).

Step 6 . Phosphate buffer (pH 6.5, 0.1 M) in sufficient quantity

to bring the column elute to pH 6.5 was passed through the columns

(required about 55 ml). EDTA, 0.2 ml of 10% sol, was also added to

the buffer and allowed to pass through the columns.

Step 7. Ten ml of purified water containing 0.1% EDTA was

passed through the columns to remove the buffer.

The columns were now ready to receive the trichloroacetic acid

extracts which were brought to pH 6 .2-6.5. After passage of the

extracts, the columns were washed with 25 ml of water containing 0.1% 45

EDTA to remove acidic (deaminated) metabolites and neutral substances.

Unchanged NE-^C was eluted with 17 ml of 1 NHC1 (discarded) followed 14 by 15 ml of 1 NHC1 which contained the unchanged NE— C . This procedure allows basic metabolites (O-methylated) to remain on the resin. There was no difference in the recoveries of the two isomers from the ion-exchange columns. The combined recoveries throughout the entire process were 72.14 - 0.76% (mean - SEM., n=ll). Values reported have been corrected for 72% recovery.

For separating O-methylated metabolites of the NE-^C isomers, m ethod of H aggendal (1962 b) w as slig h tly m odified. After w ashing the columns with 25 ml water to remove acidic or neutral impurities, columns were eluted with 33 ml of 1 NHC1 to remove all the unchanged 14 NE- C. This elute also contained a little quantity of O-methylated metabolites. Elution of O-methylated metabolites from the resin was then accomplished with 15 ml of 3 NHC1. After this procedure, no more radioactivity was eluted from the columns on further HC1 elution.

Exact recovery of O-methylated metabolites was not determined.

The 15 ml elutes containing unchanged NE-^C or the O-methyl­ ated metabolites formed from NE-^C were transferred to 2 0 ml beakers and evaporated to dryness. Residues from the air dried HC1 elutes were taken up in 1 ml of water and added to 14 ml of Aquasol (New

England Nuclear) and counted for two 20 min periods in a Packard

Tricarb Liquid Scintillation Spectrometer (Model 3375). Quenching 46 14 was monitored initially by adding a known quantity of C-benzoic acid standard to each sample and recounting the sample. Once

quenching was established, it was not redetermined each time. As

stated earlier in this thesis, the counting efficiency of the instrument

for ^C - was 76%.

Statistical Analysis

Results were analyzed by student's t-test, the t-test for paired

data and regression analysis .

Drugs and Solutions

Drugs used in this study were:

(-) -norepinephrine- (+) -bitartrate monohydrate, (+) -norepinephrine-

(+)-bitartrate, (-)-cobefrin base, (+)-cobefrin base and (+)-epineph-

rine-(+)-bitartrate (Sterling-Winthrop Research Institute, Rennselaer,

New York); (-)-epinephrine base, 6 -hydroxy dopamine hydrobromide

and (-)-d-m ethyl-p-tyrosine methyl ester, hydrochloride (Regis Chem­

ical C o., Chicago, Illinois); dopamine hydrochloride (General Biochem­

icals, Chagrin Falls, Ohio); iproniazid phosphate (Hoffman LaRoche,

Inc., Nutley, New Jersey); guanethidine hemisulfate (Ismelin) Dial with urethane, and reserpine alkaloid (Ciba Pharmaceutical Co.,

Summit, New Jersey); (-)-norepinephrine-7-^C-(+)-bitartrate,

specific activity 57.0 mCi/mM and (+)-norepinephrine-7-^C-(+)- 47 bitartrate, specific activity 21.2 mCi/mM (Amersham-Searle, Arlington

Heights, Illinois); (i)-normetanephrine-7-^H base, specific activity

5.7 Ci/mM (New England Nuclear, Boston, M ass.); cocaine hydro­ chloride (Merck Chemical Division, Rahway, New Jersey); and

3 1 ,4 1-dihydroxy-ot-methyl-propiophenone (U-0521) (Upjohn Company,

Kalamazoo, Michigan).

Reserpine was prepared as 0.5% solution in 10% ascorbic acid.

Solution of 6 -hydroxy dopamine was prepared in 0.1% sodium metabi­ sulfite on ice and gassed for at least 10 min with nitrogen. Solution of U-0521 was prepared by continuous stirring for two hours in 10% aqueous propylene glycol. Stock aqueous solutions of iproniazid and guanethidine were prepared. Solutions of isomers of unlabelled catecholamines, tropolone, oi-MPT, and U-0521 were prepared fresh daily. Working solutions of labelled (-)- and (+)-NE were prepared in

1 % sodium metabisulfite, protected from light and air and stored at

4°C. All drugs and chemicals were dissolved in demineralized double distilled water.

The signs (-)- and (+)- refer to the direction of rotation of plane polarized light, levo and dextro respectively. The sign (t) refers to the racemate compound. CHAPTER III

RESULTS

Influence of COMT inhibition on the magnitude and duration of action of catecholamines on rabbit aortic strips (oil-immersion technique).

Kalsner and Nickerson (1968 a) demonstrated that after washout of the stimulant drug, the rate of relaxation of the tissue is a valid measure of the disposition of the drug in tissue and also of the ter­ mination of action of the agonist. It was also demonstrated that oil immersion of the aortic strips for long periods had no deleterious effect on the contractile mechanism or the relaxation of the tissue.

It was desirable to confirm these findings and prove the reproducibility of this technique. Strips prepared from the same aorta were contracted by (-)-NE (10“®M) or (+)-NE (2 x 10“^M). The rates of relaxation of the tissue were followed after draining off the aqueous medium and filling the tissue chamber with mineral oil. Fig. 3 schematically illustrates the reproducibility of the magnitude and duration of action of the optical isomers of NE. The height of contraction in six aortic

+ + strips with (-) -NE was 44.4 - 3.6 mm, and 45.0 - 4.0 mm in first and second trials respectively. The corresponding heights of contraction in five aortic strips treated with (+)-NE (2 x 10“ 6 M) w ere 3 6 .8 - 4 .0 *(■ mm and 35.8 - 3.8 mm. The durations for relaxation of the strip down

48 49 RABBIT AORTA I - IPRONIAZID OIL OIL C-COCAINE P-PROPRANOLOL

n=6

217 sec 223sec WASH ±40 ±26

-)-NOREPI )-NOREPI. e IO"8M

n = 5

WASH 268 sec 2rbsec ±48 ±62

“1— / ---- I C P (+)-NOREPI. C P (+J-NOREPI. 2 x I0"6M 2 x I0 ‘ 6M

Figure 3

Schematic illustration of the magnitude and duration of the effect of optical isomers of NE on the paired rabbit aortic strips. Tissue was exposed to 10“^M iproniazid for 30 minutes and washed. The tissue contact time for 10~^M cocaine and 10“®M propranolol was 15 and 10 min respectively. The figure illustrates that magnitude and duration of the effect of both (-)- and (+)-NE are reproducible, n = number of observations. A and A 1 refer to the duration for 50% of maximal con­ traction (in sec - SEM) with (-)- and (+)-NE respectively in the first trial. B and B' refer to the corresponding values in the second trial. A'/A and B'/B refer to the ratios of duration of action of (+)- and (-)-NE in the first and second trials respectively. The oil immersion technique of Kalsner and Nickerson (1968a) was used. .50 to the 50 percent level in the first and second trials as shown in Fig.3, were 223 * 40 and 217 - 25.8 sec for (-)-NE and 268 + 47.9 and 276 ±

62 sec for (+)-NE respectively.

The influence of tropolone on the pharmacological effects of

NE isomers is illustrated in Fig. 4. The data from every pair of aortic strips were analyzed to obtain net increase in the magnitude and duration of the isomers by tropolone. The difference in contraction heights of (-)-NE before and after tropolone gave the net increase in height by tropolone. This net increase in magnitude of contraction produced by (-)-isomer was compared to that obtained from the paired tissue for the (+)-isomer. The results were not found to be statisti­ cally significant. In three out of eight experiments, the effect of

(-)-NE was potentiated more,in three experiments, the effect of both the isomers was equally potentiated, while in the remaining two experiments, the potentiation of the effect of (-)-isomer was less as compared to that of (+)-NE. Essentially the same results were obtained when this net increase was expressed as increase-percent of control.

This data is presented in Table 1 and Fig. 4.

The net increase in the duration of action of NE isomers and the net increase expressed as percent of the control are illustrated in

Table 2. Without tropolone, (+)-NE had longer duration of action than

(-)-NE in seven out of nine experiments. In the remaining two experiments, the duration of action of both isomers was,equal. RABBIT AORTA

OIL I-IPRONIAZID C-COCAINE OIL P-PROPRANOLOL

1663 sec I9 0 se c ±189 ±17 WASH

C P (-)- NOREPI. A'/A = 1.44 TROPOLONE 10" °M B'/B = 1.70 IO“ >M OIL OIL

WASH * 2 7 4 s e c 2828 sec I ± 6 0 ±382

I C P (+)-NOREPI. C P (+)-NOREPI. 2 xlO"6M 2 x IO"cM TROPOLONE lO^M

Figure 4

Schematic illustration of the magnitude and duration of action of the optical isomers of NE on eight paired aortic strips before and after tropolone (10“^M). Strips were exposed to iproniazid ( l C P ^ M ) for 30 min and washed. The tissue contact time for tropolone, cocaine (10“ 5m ) and propranolol (10“6 m ) was 20, 15 and 10 min respectively. A and A 1 refer to the duration for 50% relax atio n (in se c ± SEM) of the maximal.contraction produced by (-)- and (+)-NE respectively in the control strips.. B and B' refer to the similar values after tropolone treatment. A'/A and B'/B are the ratios of duration of action of (+)- to (-)-NE, before and after tropolone respectively. Refer to Fig. 3 and the text for further details. 52

Tropolone treatment prolonged the action of (+)-NE on every paired

tissue more than that of (-)-NE. The net increase in duration (tropolone

treated minus control) for (+)-isomer was significantly greater than

that of the (-)-isomer (P<.05). When this data was transformed into

percent of control, the difference between the duration of action of

two isomers was narrowed down and was not statistically significant.

This was due to the longer duration of action of (+)-isomer in the

control strips.

In another series of paired aortic strips, the magnitude and duration of action of (+)-NE and dopamine were compared. In the

control tissues, dopamine (2 x 10“^M) produced a slightly higher con­

traction in five out of nine aortic strips than that produced in the paired

strips by equimolar concentration of (+)-NE, whereas in the other four

strips, the contraction produced by (+)-NE was slightly higher. After

tropolone treatment, essentially the same relationship was found to hold good. There was no difference in the net or percent increase in the contraction height produced by either dopamine or (+)-NE (Table 1,

Fig. 5). It may, however, be seen (Table 2, Fig. 5) that duration of action of dopamine was longer that that of (+)-NE in the control strips.

After tropolone treatment, the duration of action of (+)-NE was pro­ longed more than that of dopamine. When these values were expressed as increase in duration-percent of control, the differences were more pronounced and were statistically significant. 53

TABLE I

INFLUENCE OF TROPOLONE ON THE HEIGHT OF CONTRACTION OF AORTIC STRIP BY CATECHOLAMINES (OIL-IMMERSION TECHNIQUE)

(c) Height of Contraction Net I n c r e a s e ^ Amine (mm) In crease In H eight (a) (b) In (Percent of Control After H eight Control) Tropolone Concentration

(-)-NE 10"8M 49.0± 4(e) 6 2 .3 -3 13.3-1 2 7.1—2

(+)-NE 2x 10~6M 47.0 ^7 58.3^6 1 1 .3 -2 2 4 .0 -4 (9) (+)-NE 2x 10"6M 32.7+3 42.8^3 1 0 . 1+ 1 33.5^5 dopam ine 2x 10"6M 34.5^3 41.4^3 6 . 8 ^ 1 20.513 (9) (-)-E pi io “ 8m 49.5±4 69.8±5 1 9 .7±2 39.7±5

(+)-Epi 3x 10 ~7M 53.0^3 69.0±3 16. 0 ^ 1 30.1±3 (8 ) (-)cobefrin io _7m 27.2-3 42.2-3 15.0-1 57.0-11

(t)cobefrin 4x10 7M 24.1±1 4 0 .7 -2 16.6-1 67.2±9 (mixture of (8 ) (-) and (+)- cobefrin 1:4)

(a) C ontrol strip s p retreated w ith ip ro n iazid (30 min) and w ash ed . Uptake in the nerve endings was blocked by cocaine 10“^M and propranolol, 10“^M was added to block E> -adrenergic receptors. Contact time for cocaine and propranolol was 15 and 10 min respectively.

(b) Contact time 20 min and added 5 min prior to cocaine.

(c) Tropolone pretreated minus control. 54

(d) Tropolone pretreated minus control x 100 co n tro l

(e) M ean - S .E .M .

Numbers in parentheses in the control column refer to the number of observations.

Strips were paired between (-)- and (+)-NE; (+)-NE and dopamine; (-)- and (+)-Epi and (-) and (±) cobefrin. RABBIT AORTA I - IPRONIAZID

OIL C - C O C A I N E

P-PROPRANOLOL OIL

n=9 WASH 2 4 2 8 (±303) sec

(+)-HOREPI TROPOLONE (+)- NOREPI 2X|0'°M IO“*M 2X10 M

OIL

OIL n=9

WASH

17 31 (±391) sec

DOPAMINE TROPOLONE DOPAMINE 2X|0‘6M Kr'M 2X10‘°M

Figure 5

Schematic illustration of the magnitude and duration of effect of (+)- NE and dopamine on nine paired aortic strips before and after tropolone (10”^M). Strips were exposed to iproniazid (10“^M) for 30 min and washed. Tissue contact time for tropolone, cocaine (10~^M) and propranolol (10”®M) was 20, 15 and 10 min respectively. Values on the horizontal lines with double headed arrows refer to the mean duration for 50% relaxation (in sec - SEM). n = number of observa­ tions. For further details, refer to Fig. 3 and the text. The results of optical isomers of Epi were also analyzed for the net and percent of increase in the magnitude and duration of action after COMT inhibition. The results are illustrated in Fig. 6 and

Tables I and II. Both the magnitude and duration of action of the isomers were almost equally potentiated by tropolone.

(+)-Isomer of cobefrin was much less active than (-)-cobefrin.

Therefore comparison between isomers of cobefrin was not possible.

The potentiation and prolongation of the effect by tropolone of (-)- cobefrin was compared with that of a mixture of 1:4 of (-)- and (+)- cobefrin. Tropolone potentiated the effects and duration of action of

(-)-cobefrin or those of mixture of cobefrin isomers equally. Results are presented in Tables I and II, but are not illustrated by a schematic d iagram . 57

TABLE 2

INFLUENCE OF TROPOLONE ON THE DURATION OF ACTION OF SOME CATECHOLAMINES ON RABBIT AORTIC STRIPS (OIL-IMMERSION TECHNIQUE)

D uration - 50% N et In crease Amine Concentration Relaxation In crease In D ura­ Control After In tion Tropolone D uration (% of Control) CO iH 1 o (-)-NE 2 190-17 1663-189 1672-197 856±155

(+)-NE 2xl0"6M 274±60 2828-382 2551-369 1105-221 (9)

(+) -NE 2x 10~6M 316-54 2428^303 2011^320 1026-299 (9) dopam ine 2x 10“ 6M 610-117 1731-391 1221-325 283-67 1 00 }_• O (-)-E pi s 124+17 1564-207 1439±196 12 85—171

(+)-Epi 3x 10_7M 156±16 1724-174 1567±165 1049-111 (8 ) l H o (-)-cobefrin 2 141—18 1397-173 1243±161 816±103

(i)-cobefrin 4xl0- M 154^19 1462-180 1307^167 869±79 (mixture of (8 ) (-)- and (+)- co b efrin , 1:4)

(a) Control strips pretreated with iproniazid (30 min) and washed. Uptake in the nerve endings was blocked by cocaine 10 ^M and propranolol, 10~®M was added to block _B_-adrenergicreceptors. Con­ tact time for cocaine and propranolol was 15 and 10 min respectively. 58

(b) Contact time 20 min and added 5 min prior to cocaine.

(c) Tropolone p retreated minus c o n tro l.

(d) Tropolone pretreated minus control x 100 control

(e) M ean - S .E .M .

Numbers in parentheses in the control column refer to the num­ ber of observations.

Strips were paired between (-)- and (+)-NE; (+)-NE and dopa­ mine; (-)- and (+)-Epi and (-) and (-) cobefrin. RABBIT AORTA 59

X - IPONIAZID C - COCAINE' P - PROFRAKOI jOL OIL

n = 8 IG35 coc WASH + 2 1 0

(-)-EPI., 10"° M A‘/A =■ 1.23 TROPOLONE OIL B'/B = 0.9b

OIL

1731 soc V/ASH + 214 r\i I60sec

(+)-EPI., 3x10 M (+) ■ EPI., 3 x I0~ M TROPOLONE

Figure 6

Schematic illustration of the magnitude and duration of effect of equiactive concentrations of Epi isomers on eight paired aortic strips before and after tropolone (10“ 4 M). The strips were exposed to iproniazid (10~^M) for 30 min and washed. The tissue contact time for tropolone, cocaine (10“^M) and propranolol was 20, 15, and 10 min respectively. Values on the horizontal lines with double headed arrows refer to the mean duration for 50% relaxation (in sec t SEM ). n = number of observations. For further details, refer to Fig. 3 and th e te x t. 60

Influence of U-0521 on the in vivo cardiovascular responses to Epi isom ers

The influence of U-0521 (lOOmg/kg) on the effects of (-)-Epi on the heart rate and blood pressure of anaesthetized, reserpinized, and bilaterally vagotomised rabbits is illustrated in Fig. 7. The effect of

U-0521 on the corresponding cardiovascular responses to (+)-Epi is shown in Fig. 8 . It appears that both the magnitude and duration of responses to (-)- as well as (+)-Epi are to some extent decreased after U-0521 treatment. These results seem to be paradoxical since

COMT inhibition should, if at all, lead to potentiation or prolongation of the effects of catecholamines. It may, however, be seen from Figs.

6 and 7 that after U-0521 treatment, the base line for the heart rate and blood pressure has shifted downward. Possibly, this diminution of the heart rate and blood pressure after U-0521 is the direct effect of the inhibitor. The area under the curves in Figs. 7 and 8 , after

U-0521 treatment, however, is the same as in control.

Similar results were obtained when the inhibiter (U-0521) was

employed in a dose of 50 mg/kg. These results obtained with this dose are not illustrated. I40r 61

o-^ o CONTROL

~ 100 a— a U-0521 TREATED n=7

j i i i i i i

£ 290

r r i

w ° . i . i J___i 2 4 6 8 TIME'(min)

Figure 7

Schematic illustration of the effect of (-)-Epi (1 jig/kg) on the blood pressure and heart rate of rabbits before and after administration of U-0521 (3' ,4'-dihydroxy-methyl propiophenone, 100 mg/kg). Rabbits were'pretreated with reserpine (3 mg/kg) 16-24 hours prior to the experiment. Animals were anaesthetized with Dial with urethane, 0.6 mg/kg (Ciba). Each point represents mean - SEM of seven observations. Refer to the text for experimental details. 160

1 4 0

120 o o CONTROL a-— a U-0521 TREATED in a? 100 n=7

8 0

6 0

2 7 0

2 5 0

1 9 0

T I M E ( m i n )

Figure 8

Schematic illustration of the effect of (+)~Epi 100 ug/kg) on the blood pressure and heart rate of rabbits before and after the administration of U-0521 (100 mg/kg). Rabbits received reserpine (3 mg/kg) 16-24 hours prior to the experiment. Animals were anaesthetized with Dial with urethane, 0.6 mg/kg i.p. (Ciba). Each point represents mean - SEM of seven observations. Refer to the text for details of the experiment. 63

Influence of MAO inhibition on the magnitude and duration of action of NE isomers on the rabbit aortic strip (oil-immersion technique)

These experiments were conducted to find out if the inhibition of

MAO has any selective influence on the potentiation or prolongation of

the effects of equieffective concentrations of NE isomers. The oil

immersion technique as described previously was employed. Repro­

ducibility of the technique was rechecked and it was found that the

experiments were highly reproducible.

Responses of the rabbit aortic strips to isomers of NE were recorded before and after iproniazid treatment as described in METHODS

AND MATERIALS. The n et and th e percent in c re a se in m agnitude and

duration of action of NE isomers was calculated from each pair of

observations and the results are shown in Fig. 9. Iproniazid treatment was not found to exhibit any selective effect on the magnitude of action

of the isomers. Duration of action of (+)-NE appeared to be prolonged

more than that of (-)-NE, if the criterion of judgment was the net

increase i.e ., the difference between iproniazid treated and control.

This index failed to take into account the difference in duration of

action of the isomers in the control strips. It may be seen from Table

3 that duration of (+)-NE action was always more than that of (-)-NE in

the control strips. The "increase in duration, percent of control" takes

care of the variability in the control strips. When this criterion was

used to assess the effect of iproniazid, the differences in the duration 64 RABBIT AORTA

T-TROPOLONE

OIL C-COCAINE

OIL P-PROPRANOLOL

n=6

WASH I 2 9 6 soc 5I4.(±68) sec I (±10)

T T T ^ '

T C P (-)-NE, IO"eM IPRONIAZID IO‘ *M OIL

OIL

n=6

WASH

1 0 7 5 ( ± 1 0 2 ) s e c 601 (±66) sec

T C P (+)-NE, 2xlO’6M T C P (+)-NE, 2xlO"6M IPRONIAZID I0"4M

Figure 9

Schematic illustration of the magnitude and duration of effect of equi- active concentrations of NE isomers on six paired aortic strips before and after iproniazid (10“^M). Tissue contact time for tropolone (lCT^M), cocaine (10- 5 M) and propranolol (10- 6 M) was 20, 15 and 10 min respec­ tively. Strips were exposed to iproniazid for 30 min and washed. Values on the horizontal lines with double headed arrows refer to the duration for 50% relaxation (in sec i SEM). n = number of observations. Refer to Fig. 3 and the text for details. 65

of effect of isomers were found to be insignificant (Table 3).

14 In vitro accumulation of (-) and (+)-NE- C by heart slices.

Slices obtained from guinea pig hearts were incubated for 5,10, and 20 min with (-) or (+)~NE-^C (100 ng/ml) to study the time sequence effect on the tissue:medium ratios of accumulation of NE-^C. Slices obtained from rabbit and cat hearts were incubated for 5, 10, 20, and

40 min with NE-^C isomers (100 ng/ml). The results are illustrated in

Fig. 10. It was observed that the tissue:medium ratio of (-) and (+)-

NE-^C was a linear function of time up to the time intervals studied in this experiment. In all subsequent experiments, the slices were incubated for 20 min. The concentration of 100 ng/ml of NE-^C was the highest used in this study for the kinetic analysis of the accumulation of NE. Since the uptake was linear at the highest concentration studied, it was presumed to be linear at lower concentrations too. Therefore time sequence analysis of accumulation was not studied at other con­ centrations . Tissue:medium ratios for the isomers did not differ in any given species nor were the differences among the species significant.

Fig. 11 illustrates the tissuermedium ratios of isomers of NE-^C when the heart slices of different species were incubated with increas­ ing concentrations of the amine. It may be seen that the tissue:medium ratios tend to decrease with increasing external amine concentration. 66

TABLE 3

INFLUENCE OF IPRONIAZID ON THE MAGNITUDE AND DURATION OF RESPONSES OF RABBIT AORTIC STRIPS TO ISOMERS OF NOREPINEPHRINE (OIL-IMMERSION TECHNIQUE)

(+) -Norepinephrine Dopamine 2x 10"6M 2x 10_6M

H eight of D uration- H eight of D uration- Contraction 50% R elax­ Contraction 50% R elax­ (mm) ation (sec) (mm) ation (sec) + (6) C o n tr o l^ 45.8-2 296 -18 46 . 6 - 6 601.3 i 56

After (b) Iproniazid 52 .1- 6 514 - 6 8 5 3 .6 - 7 1075 -101

(c) Net Increase 6 .3 ^ 1 218 1 54 7 t 1 4736 1 6 6

Increase - ... % of Control 13.3113 71.8 -15 14.4 — 1 80.0^13

(a) Strips were pretreated with tropolone 10~ M, cocaine 10~ M, propranolol 10 M to inhibit COMT, uptake in the adrenergic nerve endings and £_-adrenergic receptors for 20, 15, and 10 min respectively. Cocaine was added 5 min after tropolone but 5 min prior to propranolol.

(b) Contact time 30 min and then washed for another 30 min. Treatment with tropolone, cocaine and propranolol was also repeated as for con­ trol strip.

(c) Iproniazid pretreated minus control.

(d) Iproniazid pretreated minus control x 100 control

(e) Mean of six observations - SEM. o 67 q ------O ( - ) - N E CAT *4 DC O ------o ( + ) - N E 5 2 LUo IU ZD CO CO

6

5 O ( - ) - N E RABBIT 1 ■ O ( + ) - N E s 4 2 o LU 3 LlI 2 zd CO CO

5 Q ------O ( - ) - N E

G U I N E A P I G 4 -O ------O ( + ) - N E 3 2

0 10 20 30 40

MINUTES

Figure 10 14 Time-sequence analysis of the accumulation of (-)- and (+)-NE- C by cat, rabbit and guinea pig heart slices. Each point is the mean of four observations. Standard errors ranged from 7-18% of the mean. Bath concentration of the isomers was 100 ng/ml. Slices were preincubated with iproniazid (10 M) and tropolone (10~^M) in the bath for 30 min. Lines were drawn by regression analysis. Panels A,B, and C refer to accumulation of NE-^C isomers by heart slices from cat, rabbit, and guinea pig respectively. o

O ( — ) — N E CAT § v^0™ 0(+)-NE 2 3 8 s ui

f= 0

1 RABBIT 2 O -O (+) — NE 2 o 1U 2

Q 5 o— 0 n ' = w 1 O O (+) — NE G U I N E A P I G ~ ----- 32 o UI 2 ui

2 0 60 100 140 180

CONCENTRATION (ng/m l)

Figure 11

Accumulation of (-)- and (+)~NE-^C by cat, rabbit, and guinea pig heart slices at different concentrations of the isomers. The slices were incubated with the isomers for 20 min. Each point is a mean of at least four observations. The standard errors ranged from 5-35% of the mean. Slices were preincubated with iproniazid (10“^M) and tropolone (10~"^M) for 30 min in the bath. Tissuermedium ratios tend to decrease at higher concentrations of NE in the medium. Panels A, B, and C refer to accumulation of NE~*4C by heart slices from cat, rabbit and guinea pig respectively. 69

For each isomer, the tissuermedium ratios, at external NE concentra­ tion of 200 ng/ml, were lower than those at lower concentrations.

These results indicate that accumulation of NE-^C in the heart slices occurs by a saturable process.

14 Determination of kinetic constants of accumulation of NE- C in heart s l i c e s .

The heart slices from cat, rabbit, and guinea pig were incubated for 20 min with (-) or (+)-NE-^C in concentrations ranging from 10-100 ng/ml. Double reciprocal plots of 1/v (rate of uptake of NE, ng/gm heart tissue/20 min) v/s l/S (conc. of NE-^C, ng/ml) were plotted according to the method of Lineweaver and Burk (1934). The plots yielded straight lines with positive ordinate intercepts in all the spe­ cies (Fig. 12). The kinetic parameter, Km, is defined as concentration of NE-^C required for achieving half maximal velocity and V max, as the maximal velocity of the uptake. The parameters, Km, V max, and

V max/Km, calculated from the Lineweaver-Burk plots, are presented in Table 4. It may be seen from Fig. 12 and Table 4 that in cats and guinea pigs both Km and V max are higher for (-)-NE than for (+)-NE whereas in rabbits, the reverse was found to be true. The ratio

V max/Km for (-) or (+)-NE was not found to be significantly different in any of the species under study. 2.5

20

0.5

3 0 C' ■» M - NE O — 0 (+» ~ NE

2.5 0 * 6 -7

2.0

1.0

0.5

I/S

Figure 12

Lineweaver-Burk plots of accumulation (l/ng/20 min/g, 1/v) versus substrate concentration (1/ng/ml, 1/S) of (-)- and (+)-NE^C in cat, rabbit and guinea pig heart slices. Slices were preincubated with iproniazid (10~^M) and tropolone (10 M) for 30 min in the bath before incubation with the isomers for 20 min. n = number .of observations. Vertical bars represent SEM. Lines were drawn by regression analysis. 71

TABLE 4

KINETIC CONSTANTS FOR UPTAKE OF NE-4C ISOMERS IN HEART SLICE^ OF VARIOUS SPECIES.

Km (fa moles) V max (nq/g/min V max/Km S pecies (-) NE (+) NE R NE (+) NE (-) NE (+) NE

C at 1.00 1.79 38.45 61.50 3 8 .4 3 4 .3

Rabbit 1.12 0 .6 8 37.30 25.90 33.30 38.08

Guinea Pig 1.12 3 .6 9 34.72 96.15 31.00 26.0

Ratb 0.7 9 0.9 9 21.97 33.80 27.8 34.0

aThe slices were incubated with (-)- or (+)-NE-*4C (10-100 ng/ ml) for 20 min. b Data taken from Krell and Patil (1972 b). 72 14 Effect of guanethidine on uptake of NE- C Isomers in heart slices .

Guanethidine has been reported to inhibit uptake and storage of NE by the intraneuronal granules (Shore and Giachetti, 1966;

Lundborg and Stitzel, 1968, and Sachs, 1970). In hearts obtained from animals pretreated with guanethidine, the uptake of NE-^C should therefore be a function of neuronal membrane alone. It was therefore intended to study the uptake of NE- C14 isomers in guanethidine pre­ treated animals. The effect of guanethidine treatment on the uptake of

NE-^C has been illustrated in Fig. 13. It may be seen that treatment with guanethidine resulted in a significant and equal inhibition of uptake of both isomers of NE-^C in guinea pig and cat hearts but not in rabbit hearts.

In vivo accumulation and retention of (-)- and (+)-NE-^C in mice hearts.

Mice were infused with (-) or (+)-NE-^C and were sacrificed at different intervals of time, ranging from 15 min to 4 hours. The unchanged NE-^C content of the hearts was measured. The data was analyzed by regression analysis and the results are presented in Fig.

14. It may be seen that in the control mice, (+)-NE-^C accumulated a little more than (-)-NE initially but the (-)-isomer was retained better.

This observation is in agreement with that of Iversen et al., (1971).

Kinetic analysis of the data was performed according to the 400 CAT RABBIT GUINEA PIG 300 (-)NE-CONTROL- (-)NE-CONTROL- (-)NE-CONTROL 200 n=6-7 n=4 'n=4 fc 100 (-)NE- GUANETHONE- HNE-GUANETHIDINE- TREATED - "5 ^— (-) NE- GUANETHID1NE- TREATED TREATED

300 (+)NE-CONTROL- (+)NE-CONTROL- (+)NE-CONTROL- n = 6 -J ^ x n=4 200 5 / v (1=6-7, ^ n=4 100 ■{+)NE- GUANETHIDINE- — {+) NE- GUANETHID1NE- TREATED (+) NE-GUANETHIDINE- S - TREATED TREATED i i i ' i i i i i i J 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 l4C-NOREPINEPHRINE (ng/ml)

Figure 13

Accumulation of (-)- and (+)-NE-^C by the cat, rabbit and guinea pig heart slices in control and guanethidine treated animals with varying concentrations of the isomers. Guanethidine, 20 mg/kg, was administered i.p. 16-24 hours before experiment. All slices were incubated with iproniazid (10"^M) and tropolone (10- ^M) for 30 min in the bath before incubation with the isomers for 20 min. n = number of observations. Vertical bars denote SEM. Lines were drawn by regression analysis. 74

100

crh— < LJ X

CP n = 5 - 6 c

© © ( - ) N E I4C - c o n t r o l o— o (+)NEl4C-C0NTR0L h------a ( - ) N E,4C - IPRONIAZID AND aMPT □------□ (+) NEI4C - IPRONIAZID AND aMPT

TIME (h)

Figure 14

Semi-logarithmic plot of disappearance of (-)- and (+)-NE- C from mouse heart after an i.v. infusion of (-)- or (+)-NE- C, 40 jag/kg. n = number of observations. Vertical bars denote SEM. Lines were drawn by regression analysis. Treatment with iproniazid andc*-methyl £-tyrosine was accomplished as described in the Methods section. 75 first order rate equation. According to this equation, the concentration of a drug at a site at any given time is a function of the initial concen­ tration of the drug. This statement could be written mathematically in case of NE-^C as:

-d(NE) = k (NE) (1) dt where -d(NE) = rate of disappearance of NE with time and dt k = rate constant for disappearance of NE. Equation (1) could be rearranged to write d(NE) = -kdt (2) NE

Let NE = the initial concentration of NE, and NE. = the con- O I centration of NE at time t, integration of the equation (2) yields:

Jtn NEt = _kt (3) “NE o

Rearranging equation (3) yields equation (4), log NEt = log NEQ - Jet (4) 2.303

The rate constant k was obtained by plotting log NEt versus t. Half lives were calculated as follows. Half life of NE may be defined as the time taken for half of the NE to disappear. Substituting

NEt = 1/2 NEq and t'/a for t, equation (3) may be rewritten as:

Jin 1 / 2 NE0 = -kt 7a. (5) NE o 76

Solving equation (5) for t \ (half life), we obtain t ‘/a - 2.303 x log 2 = 0.693 /gv k k i 14 The rate constant k(hr_J-) and half lives of NE- C isomers of racemic NE obtained in the control and various drug treated animals are shown in Table 5 .

Kinetic analysis reveals that both (-) and (+)-NE-^C disappear in a single exponential phase and each with a separate rate constant.

Half lives of the isomers show that (-)-NE has a three times longer half life than that of (+)-form.

Disappearance of (+)- or (t)-NE from hearts of iproniazid treated m ice.

A considerable evidence (Giachetti and Shore, 1966b) which suggests that MAO degrades (-)-NE preferentially over (+)-NE exists.

Therefore, less (-)-NE would be available for the uptake in the heart under normal circumstances. Therefore, in all subsequent experiments,

MAO was inhibited by iproniazid, a long lasting, irreversible inhibiter of the enzyme. Results on the disappearance of (-)-, (+)-, or (±)-NE-

14C in iproniazid treated animals are illustrated in Fig. 15. It may be seen that the order of initial accumulation was (-)-NE >(+)-NE >

(l)-NE-^C. In the control mice, however, initial accumulation of

(+)-NE was a little higher as compared to that of (-)-NE. This 77 observation is in agreement with that of Giachetti and Shore (1966 b), that MAO is stereoselective.

As in the control group, the (-) and (+)-isomers disappeared in a single phase. Disappearance of (^-NE-^c, on the other hand, was found to be biphasic. The rate constants and half lives of disappearance of all the three forms are shown in Table 5. These results show that the rate of disappearance of the isomers was almost similar to that observed in the control group. The two rate constants of

(i)-NE observed in this study are quite interesting. The first rate constant is close to that of the rapidly disappearing (+)-isomer while the second rate constant resembles that of the slowly disappearing

(-)-NE. This observation clearly demonstrates that (i)-NE is a mix­ ture of two different compounds whose behavior is independent of each other and caution therefore should be exercised in using racemic NE as a tracer for the natural transmitter which is of (-)-form only. 100 O 9(-)N E l4C • 0----- 0(+)NE,4C IPRONIAZID TREATED «----- ©(±)NEI4C .

U)

20 22 24 TIME (hr)

Figure 15

Semi-logarithmic plot of disappearance of NE-^C from the mouse heart after an intravenous infusion of (-)-, (+)- or (i)-NE-*- 4 C, 40 pg/kg. Each point is a mean of 4-9 observations. Vertical bars denote SEM. All the mice received iproniazid (100 mg/kg) i.p. 16-20 hours prior to receiving NE infusion. Lines were drawn by regression analysis. Note a mono- phasic decline of (-)- or (+)-NE and a biphasic decline of (t)-NE-^ 4 C. 79

TABLE 5

RATE CONSTANTS OF DISAPPEARANCE AND HALF LIVES (t 'U) OF NE-14C ISOMERS FROM THE MOUSE HEARTS AFTER DIFFERENT TREATMENTS

e Treatment Isomer Rate Constant t 'la f (hr-1) (hrs)

Control (-) .09 7.62

(+) .26 2.57

Iproniazid 3 (-) .07 9.32

Iproniazid (+) . .17 3.92

(i) .27 2.19 Iproniazid (±) (ii) .06 1 0 .1 0

Iproniazid ) (-) .09 7.52

Guanethidine+ b ) ! (+) .15 4.57

Iproniazid ) (-) .64 1.07 + c > Reserpine ) (+) .87 0.78

Iproniazid ) (-) .08 8 . 0 0 + ^ ^ o^-MPT ) (+) .0 2 2.56 80 aIproniazid, 100 mg/kg was administered i.p. 16-20 hours before NE-l^C isomers.

T. Guanethidine, 20 mg/kg given 8-10 hours before iproniazid and 24-30 hours before NE-^C.

CReserpine, 3 mg/kg given 8-10 hours before iproniazid and 24-30 hours before NE-^C. j eat-MPT was given in 2 doses, 200 mg/kg each. The first dose was given 4 hours and second dose 1 hour before NE-^c infusion.

Rate constants (k) were calculated from the slope of the plot of NE versus time as described in RESULTS section. f t'//L= half life = .693 k 81

14 Disappearance of NE- C isomers in guanethidine or reserpine treated anim als.

Both reserpine and guanethidine are known to deplete endo­ genous NE. These drugs are known to do so by inhibiting the amine uptake mechanisms in the storage granules of the adrenergic nerves.

In studies on the uptake of ^C-NE in heart slices, described earlier, guanethidine was found to have a variable effect. These experiments were, therefore, conducted to gain more insight into the mechanisms of guanethidine action and to compare the effects of these two similarly acting drugs. The disappearance curves are illustrated in

Fig. 16 and the kinetic analysis is given in Table 5. The results demonstrate that guanethidine had very little influence on the rates of disappearance of isomers. Reserpine, on the other hand, markedly enhanced the release of both isomers and only negligible amounts of

NE were detected in the tissue four hours post-infusion. Reserpine also abolished the stereoselectivity in disappearance or storage, as observed in the control experiments. In this regard, guanethidine was without effect. 100 0----© HNE l4C - IPRONIAZID TREATED O O (+)NEhC - iproniazid treated B— B HNE'4C - IPRONIAZID AND GUANETHIDINE O —a (+)NEi4C - IPRONIAZID AND GUANETHIDINE

orh < UJ X

A A (-JNE^C - IPRONIAZID AND RESERPINE A A (+)NEI4C - IPRONIAZID AND RESERPINE

20 22 24 TIME (hr)

Figure 16 14 Semi-logarithmic plot of disappearance of NE- C from mouse heart after an i.v. infusion of (-)- or (+)-NE-^C 40 pg/kg. Mice were pretreated with either iproniazid alone or iproniazid and guanethidine or iproniazid and reserpine as described in Method and Materials. Each point is a mean of 4-9 observations. Vertical bars denote SEM. Lines were drawn by regression analysis. 83

Disappearance of NE isomers from hearts ofcH-MPT treated mice.

The drug, 2 ^-MPT is a competitive inhibiter of tyrosine

hydroxylase, the rate limiting enzyme in the biosynthesis of NE.

Treatment with this drug results in complete inhibition of synthesis

and consequently depletion of endogenous NE. In d.-MPT treated

animals, accumulation and disappearance of exogenously administered

NE-^C could thus be studied more clearly without being diluted or

exchanged with endogenous NE. Moreover, because the storage

vesicles of the adrenergic nerves are emptied by cx-MPT treatment,

this treatment is expected to enhance the uptake of exogenous amine.

If the granules exhibit stereoselectivity in uptake or storage of NE,

_cX~MPT treatment should also result in greater accumulation of

(-)-NE-^C over that of (+)-NE-^C. With these objectives, these

experiments were conducted. The disappearance curves and the kinetic

data are shown in Fig. 14 and Table 4 respectively. Fig. 14 clearly

demonstrates that CV-MPT treatment resulted in enhanced accumulation

of both isomers of NE. The accumulation of (-)-isomer, however, was

enhanced considerably more than that of (+)-form. As compared to

control, the rate constants of disappearance and half lives of the

isomers were not affected by Or-MPT treatment (Table 5). 84

Disappearance of isomers of NE-^C from the hearts of 6 -hydroxy dopamine treated mice.

It has been demonstrated that 6 -hydroxy dopamine causes a severe damage and specific degeneration of the adrenergic nerves.

Most of the adrenergically innervated tissues are chemically sympa- thectomized by this drug, and the heart is particularly sensitive

(Thoenen and Tranzer, 1968; Malmfors and Sachs, 1968). The accumulation of radioactivity in the hearts of 6 -hydroxy dopamine treated mice after an infusion of NE-^C should therefore be largely extraneuronal. Treatment of the mice with this drug, therefore, was resorted to assess the extent of NE-^C or metabolites accumulated extraneuronally in the hearts after an i-.v. infusion of NE-^C isomers.

Mice, treated with 6 -hydroxy dopamine were sacrificed 15 min and 4 hours after infusion of the isomers. The concentrations of NE-^C in the hearts at these specified times is shown in Table 6 . It may be seen that accumulation of radioactivity, 15 min post-infusion was 4-6% of the control values. When the animals were sacrificed 4 hours after infusion of NE-^C isomers, barely detectable radioactivity was observed to be accumulated in the hearts. The slight accumulation observed at 15 min represents accumulation of NE-^C by intraneuronal uptake in the surviving adrenergic nerves and also the extraneuronal accumulation of unchanged NE and its O-methylated metabolites. Thus it is clear that contribution of uptake 2 and of O-methylated metabolites 85

TABLE 6

ACCUMULATION AND RETENTION OF NE-*4C ISOMERS IN HEARTS OF 6 -HYDROXY DOPAMINE TREATED (a) MICE

Time After NE-14C (-)-NE-14C m - n e ~14c infusion ng/g heart % of control ng/g heart % of control (o) (b) 15 min 4 .9 0 l .23 4.63 6.01^ .76 6.71

4 hours . 81± . 21 1.36 .01 .0 2

(a) Treatment with 6 -hydroxy dopamine was accomplished as described in MATERIALS AND METHODS .

(b) The results are mean i SEM of 6 independent observations.

(c) Control refers to the values obtained at corresponding time points in the iproniazid treated animals. 86 was rather small and that too was observed when determinations were made shorter times post-infusion. These results justify our assump- . tion that total radioactivity represented unchanged NE-^C in MAO inhibited experiments.

Determination of O-methylated metabolites of (-) and (-i-)-NE-^C.

Mice pretreated with iproniazid were used. They were infused with either (-)- or (+)-NE-^C, 40 \ig /k g and sacrificed 10 min post­ infusion. The O-methylated metabolites were separated from the unchanged NE-^C. The O-methylated metabolites formed represented only a small fraction of the total radioactivity and the CPM were below the level of our detecting system. To circumvent this problem, five different hearts were pooled together to constitute one observation.

The results are shown in Table 6 . It may be seen that O-methylated metabolites formed from (+)-NE-^C are more than those formed from 14 (-)-NE- C. In earlier experiments, it was demonstrated that accumu­ lation of total radioactivity in the heart, 5 or 15 min after infusion of

NE-^C isomers, was equal for both the isomers. Therefore (+)-NE-^C appears to be a better substrate for COMT than (-)-NE . TABLE 7

METABOLITES . 10 MIN AFTER INTRAVENOUS INJECTION OF (-)- AND (+)-NE- C IN MOUSE HEART. 3

14 Isomer O-methylated- C Significance metabolites (cpm/g heart)

+ b (-)-NE 1500 - 221 P < .01

+ c (+)-NE 2672 - 165

aFive hearts were pooled together. b + Mean of 9 observations - SEM. cMean of 8 observations 1 SEM. CHAPTER IV

DISCUSSION

Kinetic analysis of the accumulation of (-)- and (+)-NE-^C by heart slices of various species of animals .

The in vitro technique of studying the accumulation of NE iso­ mers in the heart slices proved to be a valuable one. It has the advan­ tage of providing controlled experimental conditions, e .g ., the compo­ sition of medium, amine concentration in the medium, concentration of the inhibiters, temperature and pH. The inherent difficulties of the in vivo system such as differences in cardiac output are circumvented.

This technique suffers from certain disadvantages, too. For example, the manner of presentation of amine to the tissue in slices is different from that in in vivo system or in the isolated perfused organ. In the slices, NE from the medium has to diffuse from the superficial to the deeper layers of the cardiac muscle. Thus a concentration of gradient may be established between the superficial and the inner layers. If concentration gradients for NE exist along the way, the steady-state concentration of NE would be lower in the central portion of the slices as compared to the superficial layers. To what extent this postulated concentration gradient influences the uptake of NE and thereby obscure the stereoselectivity in the slices cannot be ascertained at present.

88 Pure (-)- and (+)-radiolabelled isomers of NE have been made

commercially available only recently. Hence many previous investiga­

tors used an indirect approach to designate the stereoselectivity of the

adrenergic neuronal membrane. Generally, the accumulation of labelled (*)-NE was determined in the presence of nonlabelled (-)- or

(+)-NE. The affinity of isomers for accumulation in the nerves has been determined on the basis of the kinetic constant Km or Ki. As

discussed in the "Statement of the Problem", neither Km nor Vmax alone could ideally define the stereoselectivity, particularly when

both these constants change. At.very low concentrations of NE, the

uptake is proportional to external amine concentration and the ratio

V max/Km acts as a better kinetic constant than Km alone. At very high NE concentrations, the uptake is not dependent upon Km at all.

At moderate concentrations, both Km and V max can influence the net uptake. In many earlier studies on uptake of catecholamines, both

these parameters have been found to be different for the isomers

(Iversen, 1963, 1965; Coyle and Snyder, 1969; Krell and Patil, 1972b).

The differences in Km and V max between the isomers in the present

studies have been noted to be in opposite directions. Thus interpre­

tation about the stereoselectivity of the process of accumulation would be just opposite when either Km or V max alone are compared.

It appears quite appropriate therefore to designate the stereoselectivity on the basis of both the parameters. The ratio Vmax/Km appears to be 90 a better criterion for comparative purposes.

For subjecting the data to Michaelis-Menton type kinetic analysis, two conditions are to be fulfilled: (i) The uptake is linear in the time interval employed, and (ii) the process is a saturable one.

Both these conditions are satisfied in the present study, (Fig. 1 and 2).

Uptake of NE into the adrenergic nerves occurs in a two stage process. Initially it involves an influx of the amine across the neuronal cell membrane, followed by incorporation of the amine into intracellular storage granules, (Carlsson et cd. , 1963; Stjarne, 1964; Iversen, 1967).

Euler and Lishajko (1963), on the basis of their studies on sympathetic nerve granules, have presented evidence that incorporation of NE into granules in the nerve terminals is an active process. Dengler et al.,

(1961, 1962) and Iversen (196 7), on the basis of kinetic studies and

Wakade and Furchgott (1968),with the aid of metabolic inhibiters, have demonstrated that initial phase of the uptake, i.e ., the influx of NE through the neuronal membrane,is also an active process. In a plot of uptake versus time at different concentrations, the linear part of the graph at the highest concentration of NE used probably represents the initial accumulation. Coyle and Snyder (1968); Draskoczy and

Trendelenburg (1968) and Iversen (1971) have indicated that the initial accumulation is mainly neuronal. Since in present experiments on slices it was observed that the uptake was a linear function of time 91 for at least 20 min at 100 ng/ml NE concentration, it is likely that most of the NE-^C accumulated in the slices was the result of the neuronal uptake.

In the light of above reasoning, two conclusions are apparent,

(i) The adrenergic neuronal membrane of the heart lacks stereoselec­ tivity. (ii) There are no species differences in the accumulation of NE by the axonal membrane of the heart. These results are in agreement with the findings with the perfused guinea pig heart (Iversen jet a l.,

1971), the perfused rabbit heart (Draskoczy and Trendelenburg, 1968), mouse atria (Sachs, 1970) and rat heart slices (Krell and Patil, 1972b) but not with perfused rat heart (Iversen, 1963; Iversen et a l., 1971).

The uptake of NE isomers by heart slices obtained from cats, rabbits and guinea pigs was also examined after guanethidine treat­ ment. Guanethidine was employed in order to block the uptake by the NE storage granules (Shore and Giachetti, 1966; Lundborg and

Stitzel, 1968). Reserpine which is also known to inhibit uptake by storage vesicles was avoided because it was reported to inhibit a sodium dependent optically specific uptake mechanism in the adrener­ gic neuronal membrane (Sugrue and Shore, 1969). In addition reserpine has been reported to induce permeability changes in the neuronal membrane of the rabbit heart slices and, thus, influence passage of the amine through the membrane (Giachetti and Shore, 1970). It was observed that guanethidine treatment significantly inhibited the 92 uptake of both isomers of NE-^C in cat and guinea pig heart slices but was ineffective in slices from the rabbit heart. The species differences are obvious. Even in species in which guanethidine was effective in inhibiting the accumulation of NE, mechanism of its action is far from clear (Maitre and Staehelin, 1971; Krell and Patil,

1972a). Guanethidine treatment, therefore, did not prove as a useful tool for understanding of the mechanisms involved in the uptake of NE.

According to Dengler et al. (1962) and Snyder et al. (1968),

MAO influences the uptake of (±)-NE- H in cat and rat brain slices at short intervals of time. Since MAO is reported to be stereospecific for (-)- isomer of NE, therefore it was inhibited by prior incubation of slices with iproniazid. Slices were also pretreated with tropolone in order to inhibit the distorting influence of COMT on uptake, if any. It appears from the evidence presented in the present study that adrener­ gic neuronal membrane of heart of various species does not discrimi­ nate between the two isomers of NE. 93 14 Retention of (-)- and (-f)-NE- C by mouse heart

Data on the retention of (-)- and (+)-NE-^C taken up by mouse heart after an intravenous infusion of (-)- or (+)-NE-^C indicate that the accumulation of both isomers by the heart was higher after 15 min than after 5 min (data not illustrated). No stereoselectivi­ ty was observed in the initial accumulation of the isomers. These results agree with those of Iversen .et al.(1971) who found no difference in the initial accumulation of (-)- or (+)-NE-^C by tissues of the mouse or by the rat heart. In the present study, even after blockade of the vesicular uptake by reserpine or by guanethidine treatment, the initial uptake of both the isomers was equal. If initial accumulation is considered as mainly neuronal (Iversen, 1971; Coyle and Snyder,

1968; DrasktSczky and Trendelenburg, 1968) the results suggest that adrenergic neuronal membrane of the mouse heart lacks stereoselecti­ vity. This interpretation is consistent with the in vitro finding in the mouse atria and irides (Sachs, 1970).

It is very clearly demonstrated in this study that the retention of (+)-NE is much less favored and it disappears much faster than

(-)-NE from the mouse heart. This finding is consistent with those observations of various other investigators on the rat heart (Kopin and

Bridgers, 1963; Beaven and Maickel, 1964; Iversen et al. ,1971). This data is also in full agreement with the suggestion that intraneuronal storage mechanisms have a lower affinity for (+)- than for (-)-NE 94 (Stjarne and Euler, 1965). The accumulation and retention of NE-^C was also manipulated by various pharmacological tools, e .g ., treat­

ment with iproniazid, reserpine, guanethidine, ^.-MPT, and 6 -hydroxy

dopamine and it was confirmed that accumulation of NE by vesicles was truly stereoselective in favor of the (-)-isomer. The following

observations in particular lent support to the above suggestions.

(i) Reserpine which is known to inhibit the uptake by the vesi­

cles abolished stereoselectivity of the retention process.

(ii) ot-MPT which depletes the vesicular stores by inhibiting the enzyme tyrosine hydroxylase in the pretreated hearts, favors the accumulation of (-)-NE-^C over that of (+)-NE-^C.

(iii) Chemical sympathectomy with 6 -hydroxy dopamine drastically reduced the accumulation of both isomers.

(iv) Twenty-four hours after the infusion of the isomers, almost all (+)-NE is lost from the heart, while considerable amount of (-)-NE- 14 C could be found in the tissue.

Results therefore favor the view that stereochemically "correct1 orientation of the beta hydroxyl group is important for the ATP-Mg - dependent process in the adrenergic nerve granules (Euler and Lishajko,

1964; Stjarne, 1964).

The process of retention described above, however, is a resultant of two separate phenomena, i .e ., storage and efflux.

The present concept of release (efflux) mechanisms in nerves is based mainly on the studies on adrenal medulla and other neuro­ secretory glands, e.g., posterior pituitary. Banks et al.(1969) reported that perfusion of adrenal medulla with ouabaine or potassium free solution increases the spontaneous and carbamylcholine-induced release of catecholamines. Both these procedures increase the intra­ cellular sodium concentration. Intracellular level of sodium is involved in regulating calcium influx into the chromaffin cells and thus the calcium-dependent secretion of catecholamines (Banks et al. ,1969).

Blaszkowski and Bogdanski (1972) also have recently presented evi­ dence that the efflux of NE from the adrenergic nerve endings in the rat heart slices is sodium and energy dependent. Thus, there is ample evidence to indicate that efflux is an active process. The similar rates of disappearance of the isomers from the hearts of reserpine treated animals indicate that the neuronal efflux from the mouse hearts may be non-stereos elective. This interpretation should however, be viewed with caution. Sugrue and Shore (1971) have reported that reserpine inhibits a sodium-dependent and optically specific amine carrier mechanism at the adrenergic neuronal membrane. The carrier mechanism could be coupled to the efflux mechanism and thus, the latter process may have been inhibited and/or its stereoselectivity obscured by reserpine treatment. 96

Norepinephrine Storage Pools

Many investigators have postulated that NE is stored in the adrenergic axons in two or more compartments with different kinetic, biochemical and pharmacological properties. This hypothesis has been based largely on the basis of the following evidence.

Potter et al. (1962) reported that tyramine even in repeated doses could not deplete more than 40% of the endogenous NE stored in the rat heart. It was subsequently demonstrated by a number of workers that even from the stores which were resistant to the effect of tyramine, electrical stimulation of the sympathetic nerves or drugs like reserpine, guanethidine, or metaraminol could evoke a significant

NE release. Potter and Axelrod (1963) postulated on the basis of avail­ able evidence that NE is stored in sympathetic nerves in at least two different pools, i.e. , "tyramine sensitive" and "tyramine resistant" pools.

Another evidence supporting the existence of multiplicity of NE storage pools within the adrenergic axons was presented by Montanari et al. (1963). These workers demonstrated that after intravenous admin- istration of (_)-4 * 3 H-NE to rats, mice and guinea pigs, the unchanged + 3 (-)- H-NE disappeared from the hearts of these animals in two phases, an initial rapid phase followed by a slow exponential phase. It was concluded that NE in nerve endings is localized in two pools, a 97 cytoplasmic and a granular pool (Montanari et a l., 1963).

Neff.et al. (1965), however, presented evidence that in the earlier studies of Potter et a l. (1962), tyramine was rapidly metabolized by MAO so the drug could not exert its full action. These workers reported that after blockade of MAO, repeated tyramine doses can deplete NE content by 90%. These observations were confirmed by

Gutman and W eil-M alherbe (1966). It was concluded by Neff et a l .

(1965) that existence of "tyramine resistant pool" was an artifact, rather than a reality.

Brodie et al* (1966) studied the disappearance of NE in various tissues of the rat after blockade of catecholamine synthesis by cx-MPT.

They reported that NE level in the heart and other organs declined in a single exponential phase. This observation is in disagreement with the hypothesis of dual-compartmental storage of NE. o Neff et §1.(1968) re-examined the disappearance of (_)- H-NE q and (-)- H-NE from rat hearts after intravenous administration of these compounds in tracer (.165 pg/kg) and non-tracer (2.16 pg/kg) doses.

They observed that after tracer doses of these compounds, cardiac

(-)-^H-NE or (1)- H-NE declined in a single exponential phase while after non-tracer doses, the disappearance of radioactivity was bi- phasic as observed by Montanari et a l . (1963). On the basis of these observations, the authors concluded that ^H-NE when given in tracer doses rapidly and uniformly mixes with the endogenous NE and 98 behaves as if it were stored in a single kinetic pool. The biphasic

3 disappearance of H-NE in the previous studies was attributed to the large doses of the amine which could have exceeded the normal intra­ neuronal storage capacity and stored in extraneuronal sites from where it would disappear at a different rate.

The results of the present study partially support the findings of Neff et al. (1968) that NE in nerve endings under normal circumstances appears to behave kinetically as a single pool. According to the present findings, the biphasic decline of (1)- H-NE in the experiments of Montanari et al.(1963) was possibly due to interference of (+)-NE with the uptake or retention of the (-)- isomer. In the present experi-

, 14 ments, (T)-NE- C was observed to decline from the mouse heart biphasically while the pure (-)- or (+)-isomers of NE-^C declined in a single exponential phase (Fig. 15). Furthermore, the rate constants of the first and the second phase of the biphasic decline of (i)-NE-^C corresponded roughly to the rate constants of decline of (+)- and (-)-

NE-^C respectively. Thus it appears that exogenously administered

NE appears to be accumulated in a single kinetic pool. Both isomers, though taken up almost equally by the neuronal membrane,differ in their retention and disappearance characteristics. Since (i) 3 H-NE is a mixture of (-)- and (+)-^H~NE, it is suggested that (i)- 3 H-NE should not be used as a valid tracer of endogenous transmitter, (-)-NE. Use of pure (-)-NE instead of the racemate is therefore recommended. 99 Stereoselectivity of COMT

The simplicity of oil immersion technique proved to be a valua­ ble method for studying the disposition of catecholamines. The results of our experiments with this technique were found to be highly repro­ ducible (Fig. 3). It was, therefore, confirmed that oil immersion per se has no deleterious effects on the tissue. The muscles could contract, relax or maintain a plateau response in oil just as well as in aqueous medium.

The contraction heights of all paired aortic strips were poten­ tiated by tropolone (Table 1). The potentiation ranged from 20-67% above the control. The differences in potentiation of action of any pair of drugs were not significant. This observation indicates that enzyme,

COMT, lacks any stereoselectivity. However, potentiation of the magnitude of action is less reliable an index of inhibiter activity as compared to the prolongation of the duration of action. The increase in duration for relaxation down to a particular level, say 50% of the initial height, reflects the inhibition of inactivation capacity of the tissue for the agonist. Kalsner and Nickerson (1969 a) observed that inhibition of both MAO and COMT diminished the capacity of the rabbit aortic strips to inactivate NE by 70-85%, yet this treatment did not result in appreciable potentiation of the effect. In contrast, they a Iso observed that cocaine clearly potentiated the responses of the tissue to NE, but it did not result in a significant increase in duration of its action 100

(Kalsner and Nickerson, 1969 c). On the basis of a number of reports,

Kalsner and Nickerson (1968, a,b ; 1969 , a ,b ,c,d ) concluded that potentiation of a effect is not a reliable index of the altered amine inactivation whereas the duration of action is. Present study is in agreement with these reports. We observed 300-1300% increase in

50% relaxation time, after tropolone whereas the augmentation of the

contraction was only slight. Thus, the interpretations based on the

potentiation of the magnitude of action are less reliable. A greater reliance has, therefore, been placed on the prolongation of the dura­ tion of action (Garg, et_a_l-, 1970, 1971).

The data presented in Table 2 confirmed the earlier in vivo observations that duration of (+)-isomers of NE and Epi and cobefrin is longer than that of the corresponding (-)-isomers in the control strips.

In the control strips, all known routes of disposition of catecholamines, but for COMT, were blocked. Longer duration of the lesser active

(+)-isomers could be explained in two possible ways: (i) COMT is stereoselective favoring (-)-isomers as its substrate, and so the (+)- isomers are relatively immune to the action of this enzyme, (ii) Longer duration of the dextro isomers is due to the higher concentrations of these compounds, used to produce an equieffective response. The present experiments support the latter possibility. It may be seen from

Fig. 4 and Table 2 that tropolone increased the duration of action of both isomers of NE, Epi, and cobefrin. Had the enzyme been 101 stereoselective, the duration of action of dextro isomers should not have been potentiated, at least not to an equal degree. The longer duration of (+)-isomers then appears to be due to more molecules of these lesser potent drugs.

On the other hand, data from the present study reveals that tropolone selectively increased the duration of action of (+)-NE as compared to its enantiomer, (-)-NE or the deoxy compound, dopamine.

Duration of Epi and cobefrin isomers was equally potentiated, however.

This observation suggests that (+)-NE is a preferred substrate for COMT.

This suggestion may seem less convincing in view of the fact that (+)-

NE is not a natural substrate of the enzyme. A few examples are, however, available where synthetic analogues are better substrates for the enzymes than the natural compounds. Hartenstein and Fridovich

(1967) observed that some of the synthetic analogues, e.g. , 2-amino-

6-chloropurine and 7-amino-thiazolo pyrimidine were better substrates than adenine, the natural substrate for the enzyme adenine aminohydro- lase. Mansoor et al. (1963) reported that 8-azoguanine is a better substrate than guanine for the enzyme guanine aminohydrolase. It could thus be possible that conformation of beta-hydroxyl group of NE plays an important part in determining the substrate specificity of

COMT. The reverse stereoselectivity of COMT may or may not be real, since it could be possible that the experimental conditions in our in vitro oil immersion system might have induced some unidentified changes. 102

Creveling et al,. (1970) reported that experimental conditions like a change in pH can cause the orientation of the catechol substrate in such a way that O-methylation may take place either at para or at meta position. In vivo, COMT metabolizes catecholamines by O-methylat- ing at the meta position only. (Axelrod, 1971).

The support to in vitro observation that (+)-NE is a better sub­ strate for COMT also comes from in vivo biochemical studies. Iversen et al. (1971) studied the formation of O-methylated metabolites after administration of equal doses of labelled isomers of NE to mice and rats. They reported that COMT lacks stereoselectivity. Their data, however, indicates that a significantly higher proportion of (+)-NE than

(-)-NE was metabolized to normetanephrine in the whole mouse, rat heart, and rat brain. In their experiments, MAO was not inhibited.

Therefore, the data could also be explained if MAO selectively oxi­ dized (-)-NE and so less (-)-NE than (+)-NE was available to be metabolized by COMT. In the present experiments, even in iproniazid treated mice, we observed that (+)-NE-^C was metabolized by COMT to a greater degree (Table 7). It appears that (+)-NE is a better sub­ strate of COMT.

In this study, stereoselectivity of COMT was also studied in vivo. The influence of U-0521, a COMT inhibiter, (Giles and Miller,

1967b,c) on the effect of Epi isomers on blood pressure and heart rate of rabbits was studied. At a first sight, the results presented in Figs. 8 and 9 look paradoxical. It appears that the cardiovascular effect of both isomers of Epi, instead of being augmented, have been diminished after U-0521 injection. A careful analysis of the data reveals that the area under the curves both before and after U-0521 is similar. The base lines for both blood pressure and heart rate had shifted downward after the injection of the inhibiter, possibly due to its toxic action. Thus the effects of Epi isomers were neither poten­ tiated nor diminished in presence of U-0521. The lack of potentiation of Epi effects by U-0521 could be attributed to a variety of causes which are discussed below.

(i) U-0521 may not be a good inhibiter of COMT. It has been used to inhibit COMT in vitro in rabbit atria (Giles and Miller, 1966,

1967a,b) and cat papillary muscle (Kaumann, 1970). It was claimed to be a better inhibiter than pyrogallol. In vivo, it has been used by

Giles and Miller (1967 c) and it was reported that it caused about 50-

75% inhibition of COMT in rat heart. The inhibition lasted for a period of 1 hour following i.p. injection of 175 mg/kg. From the above re­ ports , it appears that this compound is a moderate inhibiter of the enzyme. Since very few studies have been conducted on this com­ pound and its capacity to inhibit COMT in vivo in rabbits has not yet been demonstrated, we cannot claim that a significant inhibition of the enzyme was achieved in the present experiments. Failure to observe augmentation of Epi effects could then be attributed to 104 insufficient inhibition of COMT.

(ii) The compound, U-0521, could have exerted cardiac depres­ sant effects of its own. These effects could have obscured the poten­ tiation which was expected to result from COMT inhibition. Since toxicity studied have not been conducted on this compound, it is hard to discount the possibility.

(iii) In many earlier studies on COMT, inhibition of the enzyme has been accomplished by pyrogallol. This compound has been reported not to potentiate the effects of intravenously injected NE in cats and rats (Wylie et al., 1960) and in dogs (Crout, 1961; Izquierdo and Kau- mann, 1963). However, the potentiation of cardiovascular effects of

Epi or isoproterenol by pyrogallol has been reported in cats (Wylie et al.

1960), rats (Lembeck and Resch, 1960), mice (RosS, 1963; Ross and

Haljasma, 1964), and dogs (Izquierdo and Kaumann, 1963) . The above paradoxical effects were explained on the grounds that uptake into sympathetic nerves is more important for NE than other amines.

Isoproterenol is not taken up in the adrenergic nerves. Although Epi is taken up in the sympathetic nerves, the extent of the uptake is smaller than that of NE. The inactivation of NE by uptake is so domi­ nant that any influence of COMT is concealed. In the present experi­ ments, the uptake into adrenergic nerves was blocked by prior admin- istrrtion of cocaine. Cocaine was infused before recording the control response to Epi isomers. For recording the response to Epi isomers in 105 presence of U-0521, approximately 30 min later, a second dose of cocaine was not given assuming that blockade of uptake by cocaine would have lasted for 30 min. This assumption may or may not be valid in in vivo situation.

(iv) Failure of U-0521 to potentiate the effects of Epi in our experiments could also occur if the activity of COMT in heart and blood vessels is very low. Crout _et aH (1961) reported that activity of COMT in heart was far lower than in the liver. The endogenous levels of catecholamines in rat heart and brain were not elevated by the repeated administration of 50 mg/kg i.p. of pyrogallol every 30 min for 18 hours (Crout et a l., 1961) or after its chronic administration

(Maitre, 1966). If species differences are not too great, COMT activi­ ty in the rabbit heart would also be low. Administration of COMT inhibiter, in that case, is not expected to result in significant poten­ tiation of the effects of catecholamines. This argument seems to be less convincing in view of the potentiation of the effects of Epi and isoproterenol by pyrogallol observed by various workers. However, potentiation observed by these investigators may be more apparent than real. Gatgounis and Walton (1960) reported that catechol and related compounds, resorcinol and hydroquinone, produced cardiovas­ cular stimulant effects. Pyrogallol, a closely related compound, could too have produced the similar cardiovascular stimulant effects in earlier studies. To summarize the present experiments on the stereospecificity of COMT, it may be stated that the enzyme does not discriminate between the optical isomers of Epi and cobefrin. However, (+)-NE appears to be a better substrate for the enzyme as compared to its levo enantiomer or the deoxy derivative, dopamine. A possibility, however, exists that this observation may not be real. The activity difference between isomers of NE is quite large — about 200 fold. In the in vivo experiments on the metabolism of NE isomers in mice, (-)-NE might have caused a greater vasoconstriction and so less (-)-NE as compared to (+)-NE might have gained access to COMT sites. Therefore, the observed selectivity for (+)-NE may be apparent. The evidence from oil immersion data, however, does not support this possibility. In any event, the apparent reverse stereoselectivity of COMT for (+)-NE should be borne in mind in any further studies on catecholamines. For a clearcut answer to the problem of stereoselectivity of the enzyme, detailed kinetic analysis of the purified enzyme is required. CHAPTER V

SUMMARY AND CONCLUSIONS

1. Studies with oil immersion technique on rabbit aortic strips indicate that tropolone, an inhibiter of catechol-O-methyl transferase

(COMT) potentiated equally the effects of optical isomers of Epi and cobefrin. Tropolone, however, selectively increased the duration of action of (+)-NE as compared to (-)-NE or dopamine. These results indicate that COMT does not discriminate between the isomers of Epi and cobefrin whereas the enzyme prefers (+)-NE as its substrate.

2. Ten min after an i.v. infusion of (-)- and (+)-NE-^C, 40 pg/kg, to mice, it was found that a significantly higher proportion of

(+)-NE was metabolized to normetanephrine than that of (-)-NE in the heart. This observation supports the in vitro results that (+)-NE appears to be a better substrate of COMT than (-)-NE.

3. The cardiovascular effects of (-)-Epi, 1 pg/kg or (+)-Epi,

100 pg/kg in anaesthetized rabbits pretreated with reserpine 16-24 hours, and cocaine 5 min before the experiment, were not potentiated by U-0521, another COMT inhibiter. Failure of potentiation may be due to an unimportant role of COMT in the heart or blood vessels of rabbit or due to inefficiency of the inhibiter or both.

107 108

4. On rabbit aorta, using oil immersion technique, MAO inhibi­ ter, iproniazid was found to potentiate equally the magnitude and dura­

tion ofboth isomers of NE. Lack of selective potentiation was probably due to the lack of accessibility of NE isomers to intraneuronal MAO, since the strips were pretreated with cocaine for blocking uptake by the neuronal membrane.

5. Kinetic parameters for uptake of (-)- and (+)-NE-^C (10-100 ng/ml) were determined in the heart slices from cat, rabbit and guinea

pig, incubated for 20 min. Accumulation of both isomers was found to be linear with time up to 40 min in cat and rabbit and up to 20 min in guinea

pig. Values for Km for (-)-NE ranged from 1.00 to 1.12 p moles and that for

(+)-NE from 0.68 to 3 .69 in different species of the animals. V max values varied between 34. 72 to38.45 (ng/g/min) for (-)-NEand 25 .9 to96.15(ng/ g/min) for (+) -NE. Higher Km value in a given species for a given isomer was associated with a proportionally higher V max. The ratioV max/Km was not only the same for both isomers but was also similar in all the species.

Results indicated that there is a lack of stereoselectivity in the process of uptake of NE by the adrenergic neuronal membrane of the heart of cat, rabbit, and guinea pig and also there is a lack of species specificity.

6 . Isomers of NE-^C were infused, 40 pg/kg i.v. to normal mice and mice pretreated with various drugs. Five min after infusion, both isomers accumulated to an equal extent in hearts of normal, guanethidine treated or reserpine treated mice. This observation 109 indicates that neuronal membrane of the mouse heart is not stereoselective.

7. After i.v. infusion to normal mice, of isomers of NE-^c,

40 |ig/kg, the rate constants of disappearance (h *) of (-)- and (+)-NE were found to be .09 and .26 respectively. The corresponding half life (t '/#) values were 7.6 h and 2.5 h respectively. Twenty four hours after infusion, virtually no (+)-NE was found in the mouse heart, where­ as a considerable amount of (-)-NE-^C could still be detected. It is concluded that retention of NE by amine storing granules in the mouse heart is stereoselective favoring the (-)-isomer.

8 . In hearts of mice pretreated with reserpine, 5 mg/kg, i.p.

24-30 hours prior to i.v. infusion of NE isomers, 40 yig/kg, both (-)- and (+)-form disappeared rapidly and at a similar rate of disappearance with a t'/fl. of approximately 1 hour. These results suggest that efflux of NE from the nerves may be non-stereoselective.

9 . Mice were pretreated with C < -methyl-p-tyrosine in 2 doses of 200 mg/kg each, 4 hours and 1 hour prior to i.v. infusion of

NE isomers, 40 pg/kg. In hearts from treated mice, the accumulation of (-)-NE was increased significantly more than that of (+)-NE. Since

£L~methyl-£- tyro sine empties the storage granules by inhibiting NE synthesis, it is evident that storage granules favor the accumulation or retention or both of (-)-NE over its enantiomer. 10. Guanethidine, 20 mg/kg, pretreatment of the mice 24-30 hours before infusion of NE-^C isomers had no appreciable effect on the accumulation or disappearance of either isomer of NE by the mouse heart. It is apparent that mechanism of NE depleting action of guanethidine is dissimilar from that of reserpine. No satisfactory explanation for the lack of guanethidine effect is available.

11 . After an i.v. infusion of equal doses of (+)-, or

(i)-NE-*4C, 40 yig/kg to iproniazid, 100 mg/kg, pretreated mice, the isomers disappeared from the heart in a single exponential phase.

Racemic NE, on the other hand, declined in two phases with rate constants of disappearance (h~*) and t-k values for fast and slow phase approaching those of (+)- and (-)-NE respectively. It is concluded that the use of racemic NE for studying the characteristics of the endogenous transmitter may lead to erroneous results. Results also indicate that the exogenously administered NE is stored in a single kinetic pool. BIBLIOGRAPHY

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