The Existence of Subtypes of Alpha-Adrenergic Receptors in Canine and Rodent Vascular Smooth Muscle

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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 48106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR 77-10,634 CURRO, D.M.D., Frederick Anthony, 1943- THE EXISTENCE OF SUBTYPES OF ALPHA-ADRENERGIC RECEPTORS IN CANINE AND RODENT VASCULAR SMOOTH MUSCLE. The Ohio State University, Ph.D., 1976 Health Sciences, pharmacy

I Xerox University Microfilms, Ann Arbor, Michigan 48106

FREDERICK ANTHONY CURRO, D.M.D.

RECEPTORS IN CANINE AND RODENT

VASCULAR SMOOTH MUSCLE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Frederick Anthony Curro, B.S., D.M.D.

* * * * *

The Ohio State University

1976

Reading Committee: Approved by:

Robert W. Gardier Stanley Greenberg Popat N. Patil

Adviser Department of Pharmacology VITA

April 10, 1943 Born - Brooklyn, New York

1966 B.S., St. John's University, Jamaica, New York

1972 • D.M.D., Tufts University School of Dental Medicine, Boston, Massachusetts

RESEARCH EXPERIENCE

197A - present Postdoctoral Fellow of the National Institute of Dental Research, The Ohio State University, College of Medicine, Department of Pharmacology, and College of Dentistry, Department of Anesthesiology

1976 Visiting Fellow, University of Belgium Medical School 'at Antwerp, Antwerp, Belgium

Visiting Scientist, Boehringer Ingelheim LTD., Frankfurt En Mainz, Germany

1973 - 1974 Assistant Professor, School of Dentistry, State University of New York at Buffalo, New York

1972 - 1973 Assistant Professor, Department of Dental Materials and Anatomy, University of Texas Dental Branch at Houston, Houston, Texas

1968 - 1971 Tufts University Summer Dental Research Award, Research Assistant to Dr. Leonard Corman, Tufts University School of Medicine: Purification and Properties of Enzymes Required for the Formation of Dihydropteroic Acid

1971 Summer Research Dental Student, Harvard University under Dr. J. E. Silbert: Studies on Gylcosaminogylcans and Their Degrading Enzymes. W\ /iv PUBLICATIONS

Abstracts

Jackson, D. B., Curro, F. A., Sterling, G. H., Abrams, J. and Gardier, R. W . : The Enhancement of Nictitating Membrane: Muscarinic Contraction by Pancuronium as Antagonized by Haloperidol. The Pharmacologist, 3.1_ (2), 1975.

Greenberg, S. and Curro, F. A.: Differential Sensitivity of Portal Veins From Spontaneously Hypertensive Rats to Vasodilators. The Pharmacologist, 1S5, 1976.

Young, E. A., Curro, F. A., Ferris, T., and Greenberg, S.: Studies on the Mechanism of Amitriptyline Mediated Suppression of the Hypotensive Action of Clonidine. The Pharmacologist, 18, 1976.

Curro, F. A., Young, E. A., and Greenberg, S.: Alpha-Receptor Mediated Vasoconstriction by Serotonin: Evidence for the Possible Existence of Subtypes of Alpha Receptors. The Pharmacologist, 18, 1976.

Curro, F. A., Young, E. A. and Greenberg, S.: Amitriptyline - Mediated Enhancement of Renin Release: Suppression by Clonidine. Circulation Supplement, 1976 in press; to be presented at 49th Scientific Sessions of the AHA.

Greenberg, S. and Curro, F. A.: The Nature of the Defect in Relaxation of Venous Smooth Muscle in Hypertension. Circulation Supplement, 1976, to be presented at the 49th Scientific Sessions of the AHA.

Curro, F. A., Greenberg, S., Vanhoutte, P., and Verbeuren, T.: Venoconstriction by Serotonin is Mediated by Stimulation of a Subtype of Alpha Receptor. Federation Proceedings, 1977.

Papers in Press

Greenberg, S., Gardier, R. W., and Curro, F. A.: Differential Sensitivity of Portal Veins From Genetically Hypertensive Rats to Vasodilators. Proceedings of the Symposium on Veins, Ninth Interna tional Conference on the Microcirculation, Antwerp, Belgium, 1976.

Greenberg, S. and Curro, F. A.: Developmental Changes in Venous Smooth Muscle During the Course of Hypertension in SHR Rats. Proceedings of the Symposium on Veins, Ninth International Conference on the Microcirculation, Antwerp, Belgium, 1976.

v TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iii

VITA . iv

LIST OF TABLES x

LIST OF FIGURES • xi

INTRODUCTION . 1

Historical Concepts and Discovery of Serotonin 1

Biological Activity of Serotonin 3

Distribution and Localization of Serotonin A

Synthesis and Metabolism of Serotonin 5

Drug Receptors and the Relationship Between the Adrenergic and Serotonergic Receptors 9

Receptors for Serotonin 20

The Action of Serotonin on Smooth Muscle 26

Serotonin and the Central Nervous System 29

Problem of Thesis 33

METHODS AND MATERIALS 35

Studies on Rat Smooth Muscle 35

Effect of Serotonin and Norepinephrine on Rodent Vascular Smooth Muscle 36

Effect of Autonomic Blocking Agents on the Contractile Responses of Vascular Smooth Muscle to Serotonin, Norepinephrine and Potassium Chloride 37

\j| /vii Page

Effect of Phentolamine, Methysergide and Tolazoline on Serotonin and Norepinephrine Induced Contractions of Rat Vascular Smooth Muscle 38

Effect of Catecholamine Depletion and Blockade of Adrenergic Neuronal Reuptake on the Contractile Responses to Serotonin and Norepinephrine 39

Effect of Tolazoline on Phentolamine Induced Blockade of the Responses to Serotonin 41

Effect of Calcium Ion on the Adrenergic Blocking Activity of Phentolamine 41

Binding of 2-C^Serotonin to Rat Thoracic Aortae 42

Studies on Canine Venous Smooth Muscle 43

Contractility Studies 44

Effect of Serotonin on 7-%-Norepinephrine Efflux 45

Viability of Norepinephrine Release 46

Radioactivity Measurements 47

Statistical Analysis of Data 47

Drugs Used 48

RESULTS 49

Effect of Autonomic Blocking Agents on the Contractile Responses of Rat Arterial Smooth Muscle to Serotonin and Norepinephrine 52

Effect of Autonomic Receptor Antagonists on the Contractile Responses of Rat Vascular Smooth Muscle to Potassium Chloride 57

Blockade of Adrenergic Neuronal Reuptake and Depletion of Norepinephrine oh the Contractile Responses of Rat Vascular Smooth Muscle to Serotonin and Norepinephrine 60

Analyses of the Inhibitory Effect of Phentolamine on the Contractile Responses to Serotonin 64

viii Page

Effect of Serotonergic Receptor Blockade on the Contractile Responses of Rat Thoracic Aortae and Mesenteric Arteries to Serotonin and Norepinephrine 83

Effect of Blockade of Tolazoline Sensitive Receptors on Phentolamine and Methysergide Mediated Inhibition of the Contractile Responses of Rat Vascular Smooth Muscle to Serotonin and Norepinephrine 90

Semiquantitative Analyses of the Adrenoceptor Antagonist-Serotonin Interaction 102

Effect of Calcium Ion on Phentolamine Mediated Inhibition of Norepinephrine 103

Binding- of 2-cl^-Serotonin to Rat Thoracic Aorta 104

Effect of Phentolamine on the Contractile Responses of Venous Smooth Muscle 123

Effect of Serotonin on 7-3H-Norepinephrine Efflux 134

Effect of Methysergide on Canine Venous Responses to Serotonin and Norepinephrine 140

Effect of Alpha Adrenoceptor Antagonists on Serotonin-Induced Venoconstriction 141

Effect of Phentolamine on Serotonin Mediated ' Venoconstriction After Alpha Receptor Blockade With Tolazoline 157

DISCUSSION 167

Serotonin and Rat Vascular Smooth Muscle 167

Serotonin and Canine Saphenous Vein 181

CONCLUSION 196

LIST OF REFERENCES 197

ix 1

LIST OF TABLES

Table Page

1 Effect of Autonomic Blocking Agents on the Contractile Responses of Rat Vascular Smooth Muscle to Potassium Chloride 59

2 E° 5 0 Values of Norepinephrine and Serotonin On Rat Thoracic Aortae and Mesenteric Arteries 66

3 pA£ Values of Methysergide and Phentolamine Against Norepinephrine and Serotonin on Rat Thoracic Aortae and Mesenteric Arteries 73

4 Summary of Rat Vascular Smooth Muscle Studies 117

5 Effect of Neuronal Reuptake Blockade on the Contractile Responses of Canine Saphenous Veins to Serotonin and Norepinephrine 122

6 pA£ Values of Phentolamine and Tolazoline Against Norepinephrine and Serotonin on Canine Saphenous Vein 152

7 Summary of Canine Saphenous Vein Studies 166

8 Summary

Rat Vascular Smooth Muscle Studies 194

Canine Saphenous Vein Studies 195

x LIST OF FIGURES

Figure Page

1 Metabolic Pathways for Serotonin 8

2 Relationship Between Norepinephrine and Serotonin 19

3 Comparison of Vascular Smooth Muscle Responses to 5-HT and NE 51

4 Effect of Receptor Blocking Agents on Aortic Smooth Muscle Responses to 5-HT and NE 54

5 Effect of Receptor Blocking Agents on Mesenteric Smooth Muscle Responses to 5-HT and NE 56

6 Effect of Cocaine on Vascular Smooth Muscle Responses to Norepinephrine and Serotonin 62

7 Effect of Reserpine Treatment on Vascular Responses to 5-HT 68

8 Effect of Reserpine Pretreatment on Vascular Responses to NE 70

9 Effect of Phentolamine on Vascular Smooth Muscle Responses to Norepinephrine and Serotonin 75

10 Effect of Cocaine (5 X 10~%) on Phentolamine Mediated Inhibition of the Contractile Responses to Serotonin 77

11 Effect of Phentolamine on Responses of Vascular Smooth Muscle From Reserpine (2.5 mg/Kg i.p.) Pretreated Rats to Norepinephrine and Serotonin 79

12 Effect of Tolazoline on Vascular Smooth Muscle Responses to Norepinephrine and Serotonin 85

13 Effect on Neuronal Reuptake Blockade and Amine Depletion of Tolazoline-Mediated Inhibition of Serotonin 87

xi Figure Page

14 Effect of Tolazoline on Vascular Smooth Muscle Tone 89

15 Effect of Methysergide on Vascular Smooth Muscle Responses to Norepinephrine and Serotonin 92

16 Effect of Methysergide on Responses of Vascular Smooth Muscle From Reserpine (2.5 mg/Kg i.p.) Pretreated Rats to Norepinephrine and Serotonin 94

17 Effect of Phentolamine on the Vascular Smooth Muscle Responses to Norepinephrine and Serotonin in Rat Thoracic Aorta in the Presence of Tolazoline Plus Cocaine 97

18 Effect of Methysergide on the Vascular Smooth Muscle Responses of Rat Thoracic Aorta to Serotonin and Norepinephrine in the Presence of Tolazoline (1 X 10~^M) and Cocaine (5 X 10~%) 99

19 Temporal Responses of Tolazoline in Rat Thoracic Aorta to Norepinephrine and Serotonin in the Presence of Cocaine 101

20 Effect of Calcium Ion on Phentolamine Antagonism of Norepinephrine on Rat Aorta 106

21 Time Course of ^C-Serotonin Binding By Rat Thoracic Aorta 109

22 Effect of Phentolamine on Rat Aorta Binding of l^C-5-HT 111

23 Effect of Autonomic Agents on ^C-5-HT Uptake by Rat Thoracic Aorta 114

24 Sensitivity of Canine Saphenous Veins to Serotonin and Norepinephrine 120

25 Effect of Phentolamine on Contractile Responses of Canine Saphenous Vein to Norepinephrine and Serotonin 125

26 Phentolamine Mediated Inhibition of the Contractile Responses of Cocaine-Treated (2 X 10 M) Canine Saphenous Veins to Norepinephrine and Serotonin 128

xii Figure Page

27 Effect of Phentolamine on the Contractile Responses of Canine Saphenous Veins to Potassium Chloride 131

28 Temporal Responsiveness of Canine Saphenous Veins to Norepinephrine and Serotonin 133

29 Effect of 5-HT on Saphenous Vein Tone and % - N E Efflux 137

30 Effect of 5-HT on Saphenous Vein Tone and ^H-NE Efflux After Neuronal Reuptake Inhibition with Cocaine (1 X 10~^M) 139

31 Effect of Methysergide on the Responses to Serotonin and Norepinephrine in Canine Saphenous Vein 143

32 Effect of Methysergide on the Responses to Norepinephrine in the Presence of Phentolamine and Tolazoline 145

33 Effect of Methysergide on the Responses to Serotonin in the Presence of Phentolamine and Tolazoline 147

34 Effect of Phentolamine on the Contractile Responses of Canine Saphenous Veins to Norepinephrine and Serotonin 149

35 Effect of Tolazoline on Responses of Canine Saphenous Vein to Norepinephrine 154

36 Effect of Tolazoline on Responses of Canine Saphenous Veins to Serotonin 156

37 Temporal Responses of Canine Saphenous Vein to Norepinephrine and Serotonin in the Presence of 10-^M Tolazoline 159

38 • Effect of Tolazoline-Induced Alpha Adrenergic Receptor Blockade on the Phentolamine-Mediated Inhibition of Norepinephrine and Serotonin on Canine Saphenous Vein 161

39 Effect of Tolazoline-Induced Alpha Adrenergic Receptor Blockade on the Phentolamine-Mediated Inhibition of Norepinephrine and Serotonin in Canine Saphenous Vein 164

xiii Figure Page

40 Structural Formulas of 5-HT, NE and Antagonist 171

41 Effect of Alpha Receptor Blockade on the Responses of Canine Cerebral Vessels

xlv INTRODUCTION

Historical Concepts and Discovery of Serotonin

It has been known for over a hundred years that the vasoconstrictor activity of blood increases when blood coagulates. The suggestion was raised at that time that the unknown pressor substance released during the clotting process contributed to the increased arterial resistance of human essential hypertension. Intense interest in the mechanism of arterial hypertension stimulated the search for, and eventual isolation and characterization of, the unknown pressor substance in serum

(Page, 1968).

Ludwig and Schmidt (1868) were the first investigators to observe

that defibrinated blood increased vascular resistance in the perfused graciles muscle of the dog. Stevens and Lee (1884) made the initial observation that this was due to the appearance of a vasoconstrictor substance in clotted blood. When the clotting process was prevented by

the addition of sodium citrate to blood, Brodie (1900) found that generation of the vasoconstrictor substances was prevented. Bayless and

Ogden (1932) using defibrinated blood in the perfused kidney preparation of the dog also observed the intense vasoconstrictor activity of a

substance they termed vasotonins. An almost forgotten observation, that

ergotamine reverses the vasoconstrictor action of defibrinated blood, 2 was made by Heymans, Bouckaert and Moraes (1932) and which was confirmed

for serotonin by Page and McCubbin (1953).

In a series of papers Rapport, Green and Page (1947, 1948) describe

the isolation of a substance from blood serum which possessed two major

characteristics: 1) the substance constricted isolated perfused blood vessels and 2) the substance increased the tone of isolated intestinal

smooth muscle. Further studies by Rapport, et at (1948a) on a crystal

line complex material obtained from pig's serum showed that the vasoconstrictor substance contained an indole ring to which the

investigators gave the name serotonin.

Subsequent investigation by Rapport (1949) led to the deduction

that the active moiety of the crystalline complex was 5-hydroxytryptamine

(S-HT) complexed with creatinine and sulfuric acid. This compound,

first prepared synthetically by Hamlin and Fischer (1951) and others, proved to have all the properties of the natural serotonin (Page, 1952).

The discovery of serotonin as a normally occurring amine resulted

from independent studies on the vasoconstrictor substance. Rapport,

Green and Page, at the Cleveland Clinic, isolated serotonin from the

serum. Vialli and Erspamer (1933) in Italy gave the name enteramine to

a substance contained in extracts of rabbits' stomach mucosa causing a

color reaction after coupling with the diazonium salt of p-nitroaniline.

After an extensive investigation of the pharmacological activity and

distribution of enteramine, Erspamer (1946) had suggested that it was an

indole alkylamine. Soon after the discovery of serotonin in the blood, 3

Erspamer and Asero (1952) had established that enteramine and serotonin both had the same chemical structure.

Biological Activity of Serotonin

The serotonin present in extracts of natural materials can be quantitatively and qualitatively estimated by evaluation of the biological activity of the extract. However, serotonin shares biologic activity with at least three related indole bases, viz. tryptamine, bufotenine and the N-methyl derivative of serotonin (Lewis, 1958). The similarity of action between serotonin and tryptamine was first recognized by Reid and Rand (1951). The biological action of tryptamine was very similar to that of serotonin, with the exception that tryptamine failed to release adrenalin when injected into the arterial supply of the adrenal glands of the cat.

The identity of the synthetic serotonin and the naturally occurring material, as determined by their pharmacological reactions, was demonstrated by Page (1952). Page noted that the vascular responses to serotonin is highly variable, and that there is a need to always indicate the conditions under which responses are elicited. He described the term amphibaria, since serotonin is both a pressor and a depressor substance, but may also be only one or the other. The usual response in dogs anesthetized with pentobarbital to an intravenous injection of 0.06 to 0.12 mg of serotonin is a) an initial quick fall in arterial pressure with a bradycardia, followed immediately by b) a sustained pressor response followed by c) a prolonged depressor response 4 of lesser magnitude than the pressor response (Page, 1954). The above response to serotonin describes the one most often seen.

The response to serotonin as a pressor or depressor agent in the intact body depends upon the degree of existing neurogenic vasoconstric tor tone. Elimination of the vasocontrictor tone by ganglionic blockade or spinal cord section converts the depressor response to serotonin to a pressor response (Page, 1954).

Distribution and Localization of Serotonin

Indolealkylamines, including serotonin, occur widely in nature.

The highest concentrations of both serotonin and norepinephrine are found in phyletically older structures, which indicates their associa tion with autonomic functions and the integration of emotional patterns

(Pscheidt, et at, 1964). About 90 percent of the serotonin present in the body, which in an adult human probably amounts to 10 mg., is lodged in the gastrointestinal tract, mainly in the enterochromaffin cells and enterochromaffin-like cells (Goodman and Gillman, 1975). The remaining serotonin, about 8 to 10 percent, resides in platelets and only 1 to 2 percent within the central nervous system (Cooper, et al, 1974). In mammals, a high concentration is also localized in the pineal gland and in such animals as rats and mice, serotonin is found in the mast cell.

Serotonin is not found in mast cells- (normal or neoplastic) in man and dog, however, serotonin is present in mast cells occurring in human carcinoid tumors. The biosynthesis of serotonin from tryptophan in the enterochromaf-

fin cells has been established by the extensive investigation of

carcinoidosis. Tryptophan administered by the oral route to these

patients is followed by the elimination of labeled 5-HTP, 5-HT and

5-HIA (Figure 1). These findings strongly suggest that in the normal

intestinal tissue serotonin is synthesized and stored in the entero

chromaf fin cells. However, 5-HTP has not been detected in normal

intestine, probably because it is rapidly decarboxylated. This suggests

the difficulty in assigning from normal intestine a transport mechanism

able to carry 5-HTP to tissues where it can be decarboxylated. It is

still to be established if serotonin is present in plasma in a free form because it is difficult to avoid the disruption of platelets during the manipulation of the blood.

Synthesis and Metabolism of Serotonin

The initial step in the production of serotonin appears to be the

uptake of the amino acid tryptophan which is the primary substrate for

the synthesis. Plasma tryptophan arises primarily from the diet, and

elimination of dietary tryptophan can profoundly lower the levels of

brain serotonin. In addition, an active uptake process is known to

faciliate the entry of tryptophan into cells, and this entry site can

be competed for by certain other amino acids, such as phenylalanine.

Hydroxylation at the five position is the next step to form

5-hydroxytryptophan (5-HTP) by the enzyme tryptophan hydroxylase, which 6 is a rate limiting reaction. 5-hydroxytryptophan is then decarboxylated by aromatic L-amino acids decarboxylase to yield ,5-hydroxytamine (5-HT)

(Figure 1).

Serotonin is primarily oxidatively deaminated by the enzyme monoamine oxidase (MAO) in its process of being metabolized. The product of this reaction, 5-hydroxyindoleacetaldehyde can be further oxidized to 5-hydroxyindoleacetic acid (5-HIAA) by the enzyme aldehyde dehydrogenase or reduced to 5-hydroxytryptophol (5-HTOL) by the enzyme aldehyde reductase, depending on the NAD+/NADH ratio in the tissue

(Figure 1).

The three enzymes are present in liver and various tissues containing serotonin, including the brain. The principal metabolite,

5-HIAA, is excreted in the urine, along with much smaller amounts of

5-HTOL, mainly as the glucuronide or sulfate (Goodman and Gilman, 1975). 7

FIGURE 1.'

METABOLIC PATHWAYS FOR SEROTONIN

1. Tryptophane-5-hydroxylase present in rat intestine, toad, and Chromobaoterium violaoeum 2. 5-Hydroxytryptophan decarboxylase 3. Monoamine oxidase 4. Formation of a brown pigment 5. Aldehyde dehydrogenase 6. Glycine conjugation 7. Glucuronic conjugation of 5-HIA 8. Sulphonic conjugation of 5-HIA 9. Hydroxyindole-O-methyltransferase 10. Sulphonic conjugation of 5-HT 11. Glucuronic conjugation of 5-HT 12. Under action of ultraviolet light 13. Reaction suggested 14. Nonenzymatic complex with DPN 15. Caeruloplasmin 16. N-Acetylation 17. Hydroxylation of melatonin 18. Phenoloxidase present in Harding-Passey melanoma 19. N-Methylation present in invertebrates and in rabbit lung. N, N-Dimethyltryptamine is present in seeds of Piptadenia peregrina 20. Methylation occurring in Phalaris aurundinaoea 21. Oxidation by haemoglobin 22. Violacein 23. Produced by Amanita phalloides 24. Dihydroxytryptophan and dihydroxytryptamine 25. Transamination 26. Methylation occurring in Phalaris aurundinaoea 27. Decarboxylation

(Garattini and Valzelli, 1965)

Other than 0-methylation and deamination of serotonin, no other metabolic pathways are known as yet in vascular smooth muscle. OCHTOROBUFOTCNINE O-SUIPKATE

BurOTHIONINC

— mm* k o V/ n J COOH UNKNOWN V N «,t-om ro«oxYi*Y'TonuK PROOOCT5 OEHYOKCSUrOTEMlMg

9-HTOKOXtlBSOLC PTAUv Tc ACIQ _

t»HTOtt

IMlMt

PHENOLOXIOASE

9'HVOPOXYINOOIC ACCTALOCHTPC :Mr C M |-IW -C -C M , C*,Oj - 0-* IfmTU-S-MYDMXY- TRYMAMINC

CH,Oi

&»m c th o «t Tb t p t a m ih c

,CM,COOH

S-NrOOOKTMCUTDWIB

COMPLEX WITH OPN

W H A T t k ------10- SULPHATE "C cH -su . HO/ H t-NVDRSXVlNOOlC KTOUIICHINAMINC

FIGURE 1. METABOLIC PATHWAYS FOR SEROTONIN 9

Drug Receptors and the Relationship Between the

Adrenergic and Serotonergic Receptors

In order for an agonist to elicit its pharmacologic response in smooth muscle, it is presumed that it must first interact with its receptor on or in the cell membrane. This interaction supposedly accounts for the quantitative relationship between the response to an agonist and the dose or concentration of the agonist used. Receptors are thought of as specific molecular sites or structures in (or on) an effector cell with which molecules of a specific agonist must react in order to elicit the characteristic response of the cell to the agonist

(Furchgott, 1964). Other sites may exist within a cell which bind molecules of the agonist, and some of these sites may have specialized functions (e.g., enzymatic degradation, storage, or transport of the agonist) which could modify the concentrations of agonist reaching their primary receptors. These secondary binding sites for a drug have been termed drug acceptors (Fastier, 1964).

Qualitative differences in the pharmacological properties of com pounds which are closely related chemically may be accounted for by supposing that the compounds may differ: (a) in affinity for drug receptors which are not all of the same type, and (b) in the efficacy of their interaction with a particular receptor type. The quantitative differences may be related to the availability of the drug at the sites where its action is thought to occur. The amount of drug present at these sites will be less at any time than the amount administered. It 10

Could be considerably less if the drug does not pass readily through the cell membrane, if it is highly vulnerable to enzymatic attack, or if it has an affinity for sites which do not cause a response. The latter group are called silent receptors or spare receptors (Goldstein, et al, 1974).

The concept and experimental evidence for two distinct classes of adrenoceptors dates back to the classical study of the influence of ergot alkaloids on the effects of adrenaline and sympathetic nerve stimulation first performed by Dale (1906). The early works of Dale

(Barger and Dale, 1910) introduced two methods for differentiating types of adrenoceptors: (1) that of selective blockade by antagonists and

(2) that of comparing the relative potencies of a series of adrenergic agonists. In 1948 Ahlquist performed a classical study differentiating receptors on the basis of the order of potency of a series of sympatho mimetic amines in producing responses in each of a variety of sympathe tically innervated effector organs. The amines used by Ahlquist were

(1) epinephrine, (2) norepinephrine, (3) a-methyl-norepinephrine, (4) a-methyl-epinephrine, and (5) isoproterenol. Ahlquist tested these amines in intact dogs, cats, and rabbits and on isolated tissues from these and other species. He found the relative potencies for producing excitation of the smooth muscles of the peripheral blood vessels, i.e., vasoconstriction, to be in descending order 1>, 2>, 3>, 4>, 5. The order for producing inhibition of the smooth muscles of blood vessels, i.e., vasodilation was found to be 5>, 1>, 4>, 3>, 2. The two distinct orders of potencies led him to conclude that these were "two distinct types" of 11 adrenoceptors mediating the responses which he had observed. He proposed calling the type associated with the first order alpha and that with the second order beta. With the introduction of the adrenergic blocking agents, the concept for two major types of receptors was greatly strengthened and eventually came into general acceptance.

In 1967, Furchgott attempted to refine and extend the studies performed by Ahlquist. His conclusions were based on both the relative potencies of selected adrenergic agonists, in producing specific responses in isolated tissues from rabbit, and guinea pig, and quantita tive determinations of the affinities of specific competitive antagonists for the receptors (pronethalol for 3-receptors and phentolamine for a-receptors). He concluded that in the limited number of tissues studied there was only one type of a-receptor, but at least three types of 3-receptors. Thereafter, based on the results of the relative poten cies of a large series of sympathomimetic amines in producing responses in both isolated tissues and in intact animals, Lands, et at, (1967a, b) proposed, on the basis of statistical comparisons of the potency series obtained for different responses, two different types of 3-receptors— which they termed 3i and 32* Thus, the basis for delineating and refining the concept of a and 3 receptors is a pharmacological charac terization of responses mediated by the receptors or a quantitative determination of the affinities of specific competitive antagonists for the receptors (pAx ). 12

The definition of an a-receptor (Ahlquist, 1966; Furchgott, 1967) used In this dissertation is as follows: an a-receptor is one which mediates a response pharmacologically characterized by: (1) a relative potency series in which epinephrine > or = norepinephrine > phenylephrine

> isoproterenol, and (2) a susceptibility to specific blockade by phentolamine, dibenamine or phenoxybenzamine at relatively low concen trations. This definition is the most widely accepted for an a-receptor.

The following study employs both a characterization of responses medi ated by the receptors activated or occupied and a determination of the affinity of specific competitive antagonists for these receptors (pA2 ).

The classes of agents which are recognized at present, as being potent in antagonizing one or another characteristic pharmacological action of serotonin, encompass compounds with different chemical struc tures. Blocking agents of serotonin belong to several different classes of organic compounds, which include indolealyklamines, lysergic acid derivatives, beta haloethylamines and phenothiazines. The structural difference of the blocking molecules of serotonin is somewhat similar to the situation existing with antagonists of epinephrine. These also are highly different chemically and include benzodioxanes, beta haloethyl amines, substituted ethylenediamines, ergot and yohimbine type alkaloids.

The existence of several classes of blocking agents to sympathomimetic amines and serotonin and the fact that often members of the same class of blocking agents are effective against both types of biogenic amines raises the suspicion that the tissue receptor types of these amines might also be somewhat similar (Gyermek, 1966). Added evidence in 13 support of this came from Mansour (1957) and Innes (1962) when they suggested that d-amphetamine, a member of the sympathomimetic amine family, was effective on serotonin receptors and thus should rather be considered as a serotonin-like agent. Overlap in the substrate specificity of the metabolizing enzymes of these two classes of agents

(i.e., amino oxidase and O-methyltransferase) also suggest that sero tonin and sympathomimetic amines may be fairly close in their basic mechanism -of action. The catalyzing effect of serotonin on the formation of cyclic adenosine 3', 5 '-phosphate and on phosphorylase activity in tissue particles from the liver fluke which is similar to the effect of epinephrine in other organisms (Mansour, et at, 1960) seems to substanti ate this hypothesis further. Specie differences and the predominant tissue specificity to one type of amine, does not necessarily contradict the theory that the basic biochemical mechanisms in the action of sympathomimetic amines and indolealkylamines might be similar in nature.

Studies by Vane (1959) and Barlow (1961), designed to differentiate the serotonin receptors in peripheral organs, utilized structural analogues of 5-HT, 5-HT antagonists and monomine oxidase (MAO) inhibitors. It was observed that the inhibitory actions of BOL (2-bromo-lysergic acid diethylamide) and indole type antagonists, against serotonin and tryptamine, possessed a differential mode of action. This difference was thought to be due possibly to a selective interference with the transport of tryptamine through the cell membranes, assuming similar intracellular receptors for tryptamine and serotonin, or to different receptors entirely for these two amines (Barlow, 1961). Employing various 14 analogues of serotonin, Vane (1959) classified these agents into three different types: (1) Compounds similar to serotonin and displaying a certain affinity for tissue receptors and for MAO in the rat stomach.

However, the action of this group could not be potentiated in the isolated organ preparation by MAO inhibition; (2) Compounds similar to

5-methyltryptamine and exhibiting affinity for the receptors and for

MAO. The action of this group could be potentiated in the isolated organ by MAO inhibitors; (3) Agents similar to 5-hydroxy-l-methyltryptamine which showed affinity for the tissue receptors only and not for MAO.

Thus MAO or isoenzymes of MAO may be stored in different locations or in an area not readily accessable to all the receptors for enzymatic degrada tion. Alternatively, MAO may be more selective than the receptor for which chemical structure it can interact with to alter the responses in rat stomach. However, MAO has two forms with different substrate require ments (Jarrott, 1971); type A deaminates tyramine, norepinephrine, and

5-HT, whereas type B has little effect on either 5-HT or norepinephrine.

These observations also suggest that the tissue receptors for serotonin may be separated from the intracellular enzyme receptors.

Some of the compounds, such as tryptamine, penetrate into the cells and can be metabolized by MAO, but some, like serotonin, cannot penetrate into the cells. Studies on the dissociation constants of the active groups of serotonin and on the partition coefficients of tryptamine and serotonin, showed that serotonin did not penetrate into the lipid phase, whereas tryptamine did. The demonstration by Vane (1959) and

Barlow (1961) of different types of serotonin tissue receptors and MAO 15 receptors is important in comparing serotonin with norepinephrine because it has been suggested that the tissue receptors for norepineph rine are also responsible for its enzymatic destruction.

Besides the identity of tissue receptors and enzyme receptors for norepinephrine, the identity of certain tissue receptors sensitive to serotonin and sympathomimetic amines is still an open question

(Gyermek, 1966). In contrast to several studies which indicated that serotonin acts on specific receptor types distinguishable from those of acetylcholine, histamine and sympathomimetic amines, Mansour (1957) found in the liver fluke that serotonin and amphetamine are indistin guishable in their actions, and Innes (1962) demonstrated that on the cat spleen, serotonin and epinephrine act on the same receptor. This conclu sion was based on a thorough study using antagonists such as phenoxybenzamine, BOL and dihydroergotamine which blocked both amines. Epinephrine overdosage protected the tissue from the blocking effect of phenoxybenzamine against serotonin and epinephrine. Cross tachyphylaxis, i.e., a decrease in the response, was also demonstrated between serotonin, epinephrine and tyramine when either agent was employed to initiate the response. The situation is somewhat complicated, however, due to the possible existence of a dual action of serotonin on this organ. Based on the failure of morphine and atropine to inhibit serotonin on this prepara tion, the above study also indicates that the cat spleen is deficient in

"M" receptors. Results obtained with other antagonists suggest that the receptors of this preparation, although resembling those of the "D" receptors of the guinea pig ileum, due to their sensitivity to epinephrine, are not specific receptors for serotonin. Blockade of the pressor 16 responses of serotonin by phentolamine and potentiation by the MAO inhibitor iproniazid, in the intact rat, suggest an adrenergic mechanism in the pressor action of serotonin (Beleslin and Varagic, 1960).

This observation seems in accord with the findings of Innes (1962) in the cat.

Alpha-adrenergic antagonists have been shown to also block non- adrenergic receptors (Furchgott, 1955). In rabbit aortic strips phento lamine, as a competitive antagonist, has about one-tenth the affinity for the 5-hydroxytryptamine receptor as for the a-receptor; and dihydroergotamine actually has a higher affinity for the serotonin receptor. In addition, dibenamine and phenoxybenzamine irreversibly block the serotonin receptor as readily as the a-receptor, and in suf ficient concentrations, also irreversibly block the histamine and acetylcholine receptors. Thus, it is difficult to discern any specifi city from these compounds in blocking a specific receptor.

In general, the concomitant blockade of a non-adrenergic receptor and an adrenoceptor by an adrenergic antagonist would not be expected to influence the results of an experiment in which the antagonist was being used to characterize the adrenoceptor. However, if the basal functional state of the tissue preparation used were partly determined by the action of either an endogenous or exogenous agonist acting on the non-adrenergic receptor, then blockade of that receptor could lead to erroneous results

(Furchgott, 1972). Jequier, et alt (1969) have suggested that in the rat brain stem 5-hydroxytrytophan inhibits tyrosine hydroxylase and that 17 norepinephrine inhibits tryptophan hydroxylase. This led Zhelyaskov, et at, (1968) to postulate that the synthesis of the catecholamines and serotonin may have a possible mutual regulatory affect on each of the biogenic amines (Figure 2). 18

FIGURE 2.

RELATIONSHIP BETWEEN NOREPINEPHRINE AND SEROTONIN

Norepinephrine and serotonin may play a role in the regulation of each amine. Feedback inhibition by norepinephrine and 5-hydroxytryptophan can possibly affect the rate limiting reaction in the synthesis of serotonin and norepinephrine. ^ y - y C ° O H COOH aJ nhz nh2 H Inhibition tyrosine I inhibition iriri iui l iuii tryptophan I----- tyrosine I hydroxylase ---- 7 nhibition p— — — — > tryptophan j hydroxylase H°vT^Ssr^NV'C00H 1 i COOH H O XJ DOPA

DOPA decarboxylase 5-hydroxytryptophan H0YiO 5-hydroxytryptophan decarboxylase HOAJ NH* dopamine

dopamir 8-hydroxylase NH2 'OH

5-hydroxytryptamine (serotonin) NH 2

norepinephrine

FIGURE 2. RELATIONSHIP BETWEEN NOREPINEPHRINE AND SEROTONIN Receptors for Serotonin

The characterization of reactive sites in tissues which exhibit specific sensitivity to serotonin has been attempted using a variety of methods and approaches. Among these are the utilization of structural analogues and antagonists of serotonin and the use of monoamine oxidase inhibitors. However, it is still difficult to draw any uniform con clusions with regard to the exact nature of the serotonergic receptors

CGyermek, 1966). Studies comparing the pharmacological actions of

serotonin in vivo and in vitro with that of agents like epinephrine, norepinephrine, acetylcholine, histamine and some physiologically potent peptides, indicated that serotonin has some characteristic actions which differ from the above mentioned compounds (Page, 1953, 1958;

Meier, et al, 1957; Brownlee and Johnson, 1963). Thus it may seem justified to accept the existence of tissue receptors which preferential

ly interact with serotonin as compared to other physiologically occurring vasoactive agents. However, from the above studies, the difference in

the actions of the agents used were observed in the whole animal. Whole

animal studies neither suggest nor prove the possible existence of

pharmacologically distinct receptor sites for serotonin, since they are

difficult to interpret. The proportion of receptors sensitive to vaso

active substances other than serotonin,- which might be activated

indirectly by serotonin, are unknown. For example, serotonin may release

epinephrine from the adrenal medulla of the dog (Eble, et al> 1972) and

cat (Reid, 1952). 21

Thus, most of the studies investigating the nature of the serotonin receptor employed isolated organs and in particular, the rat uterus and the guinea pig ileum. Isolated organ systems offer the advantage of directly observing the response to various drugs, which in these studies, would be contraction. In addition, the differentia tion of the hypothetical receptor sites can usually be obtained by the application of "selective" blocking agents.

Rocha E. Silva, et al, (1953) using guinea pig ileum demonstrated that serotonin acts on receptors distinguishable from those which are stimulated by acetylcholine, histamine and bradykinin. Further testing by Gaddum and Hameed (1954) of a number of antagonists on different organs led to the assumption that even in a "simple, isolated organ like a piece of gut", more than one serotonin receptor type existed. Soon after,

Gaddum and Picarelli (1957) found that the guinea pig ileum may contain two types of serotonin receptors. Differentiation between them was made by the use of different antagonists. The findings indicated that one type of receptor of the guinea pig ileum is the nerve tissue element

(M-receptor) which is stimulated by serotonin and blocked by morphine, methadone, atropine and cocaine. The second receptor is located in the smooth muscle (D-receptor). The D-receptor, besides being sensitive to serotonin, was blocked by dibenzyline, ergot derivatives, and 5-benzyl- oxygramine. While in the case of the "D" receptors a more direct action on the contractile elements is assumed, the tissue response to stimulation of "M" receptors may involve more intermediate steps, including the 22 participation of other receptors, which eventually caused a release of acetylcholine.

It is uncertain at the present time whether: (1) two receptors for serotonin exist in this preparation, (2 ) the receptor resides in or on the smooth muscle cell membrane, (3) the receptor is an intramural neuron and part of the nervous element (Harry, 1963), (4) both types of receptors are in the nervous elements (Day and Vane, 1963), (5) these are not nicotinic receptors but differ from the smooth muscle, D receptors,

(6 ) serotonin acts on specific receptors at the intramural parasympathe tic ganglion cells (Brownlee and Johnson, 1963), and (7) some part of the action of serotonin is mediated by the release of acetylcholine.

Innes and Kohli (1969) have shown with receptor protection studies employing phenoxybenzamine in guinea pig ileum that serotonin protects against itself but does not protect against acetylcholine. This would indicate that some part of the action of serotonin is not mediated by the release of acetylcholine in this preparation and that the receptor may be located on the muscle cell.

Based on the findings outlined above, it seems that in organs like the guinea pig ileum, where both nervous and smooth muscle elements exist which are sensitive to serotonin, a priming effect on the nervous recep tors may be necessary to obtain a sufficient degree of responsiveness of the muscular receptors (Gyermek, 1966). Similar observations were made on the dog bladder (Gyermek, 1962) where two effects of serotonin could be distinguished: a fast twitch-like response which was due to the 23 stimulation of nervous receptors, and a slower contraction as a result of stimulating the smooth muscle receptors. The slower contraction, due to the muscular component was more dependent on the stimulation of the nervous elements. However, consideration must be given to the difficulty in separating the various sites of actions of serotonin on the guinea pig ileum. Thickness of preparation, length of tissue, presence of other vasoactive agents, the presence of ganglia, and the distance from the adrenergic nerve terminal to the smooth muscle cells are inherent considerations in any isolated nerve-muscle preparation.

Greenberg (I9603) reported that serotonin, on the heart of the mollusc, Venus mercenaria, has two effects. In low concentrations serotonin produces a positive inotropic effect whereas in high concen trations there is a large increase of diastolic tension. BOL (2-bromo- lysergic acid diethylamide) is effective only in antagonizing the effect of moderate doses to serotonin. Large doses of serotonin produce tachyphylaxis and also render the tissue insensitive to related indoleaklylamines, e.g., tryptamine and bufotenine. These indole com pounds and 5 -hydroxy-a-methyltryptamine in turn desensitize the heart to serotonin. The desensitizing effect is not shown against catecholamines indicating different receptor sites for serotonin and the indole com pounds. Further studies by Greenberg (1960^) on the heart of Venus mercenaria (chosen for its high sensitivity to serotonin and because serotonin is a neurotransmitter substance in molluscan hearts) indicated that not only serotonin and some indolealkylamines but also tyramine and phenylethylamine also act on the same receptors. This finding and the observation that certain indoles (those lacking an alkylamine side chain at C-3) are inactive suggest that the indole nucleus is not necessary for a serotonin-like action in the mollusc. However, one is cautioned in equating serotonin receptors in

Venus mercenaria with those of mammalian organs. While tryptamine and several N-alkylated tryptamines were found to be about 5-10 times less potent than serotonin, bufotenine proved to be about 30 times more effective than serotonin on the molar basis indicating a higher degree of specificity of the venus heart receptor to bufotenine than in the majority of mammalian receptors, where the optimal structure seems to be serotonin (Greenberg, 1960^). Furthermore, Reite (1969a, b, 1970) in a study of the evolution of the vascular smooth muscle responses to serotonin observed that only a stimulatory action is present in fish, whereas, a further evolutionary step is reached by the appearance of the inhibitory action of serotonin in amphibian. In addition, a dual response pattern which prevails in mammals (inhibitory as well as stimulatory) characterizes the reptilian vascular system. Thus, there appears to be a phylogenesis of the serotonin receptor.

The nature of the serotonergic receptor is still under scientific investigation. It appears to react with many different compounds.

Studies are complicated by the presence of other endogenous vasoactive substances which may modify or alter the responses of the serotonin receptor. There appears to be specie differences to the responses of various serotonin analogues and to serotonin itself. One major technical )

25 problem of any receptor study is to obtain the simplest possible isolated system which behaves similar to the intact organism.

Isolated organs are still very complex systems, which contain many

types of receptors, which may alter or influence the responses of the

receptor under investigation. Other factors which may influence the hypothetical interaction of agonist-receptor-antagonist are: (1 ) the

association of agonist and antagonist to the receptor is usually not a

linear function and is also reversible, (2 ) the receptor, while reacting with either the agonist or antagonist, is by its nature, liable to pos

sible morphologic change, (3) the association of the antagonist to the

receptor is a time dependent phenomenon, (4) the possibility of differ

ent time effect relationships at the different receptors, and (5) the

number and distribution of the various types of sites which influence

the responses of the agonist to its receptor. In addition, even if a

blocking agent interfers with an agonist at only one type of receptor,

two effects may take place: (1 ) competition with the agonist for the

unoccupied receptor sites resulting in interference with its fixation

to the receptor, and (2 ) attacking the occupied sites which result in

replacement and release of the agonist molecules from those sites

(Woolley and Edelman, 1958). Thus, there exists the possibility for a

very variable state and lack of equilibrium between the agonist and

antagonist in many systems due to the possible movement of serotonin

from one receptor to another. This problem is hopefully minimized in these

studies by the use of a three-hour equilibration period in which time the

vascular strips become stable and responses reproducible with time. Serotonin and 5-HT antagonists possess unique characteristics which can possibly complicate, in addition to the above mentioned factors, the agonist-receptor-antagonist interaction. These compounds often have the ability to influence the response in the following manner: (1) Serotonin often has a dual action, e.g., stimulation, and especially in higher concentrations, auto-inhibition. (2) Serotonin shows the phenomenon of tachyphylaxis. This results in a self-blocking effect by applying a series of doses which seem exclusively stimulant at the beginning of application. (3) There are serotonin antagonists which in addition to their primarily blocking responses are stimulants. This may occur either at the same receptor sites where serotonin is acting or at pseudo receptors of serotonin, the stimulation of which may somehow interfere with the action of serotonin (Gyermek, 1966).

The Action of Serotonin on Smooth Muscle

Histochemical and electron microscopic studies have shown that the morphology of the adrenergic innervation differs from organ to organ.

There are at least three important differences: (1) the density of innervation, (2) the symmetry of innervation, and (3) the distance from nerve endings to effector cells (Trendelenburg, 1972). Serotonin is thought to exert its effect on the smooth muscle cell membrane which results in an increased permeability and ionic movement altering the transmembrane potential. A membrane action of serotonin resulting in calcium entry is probably responsible for smooth muscle contraction.

Serotonin does not evoke secretion or smooth muscle contraction in the absence of calcium (Goodman and Gilman, 1975). 27.

In stlmulus-secretion coupling (Douglas, 1968) and in excitation- contraction coupling mechanisms involved in smooth muscle (Somlyo and

Somlyo, 1970), the influx of calcium ions may result from a direct action of serotonin on the membrane which increases the permeability to calcium, thereby allowing it to run passively down its electrochemical gradient. Depolarization, brought about mainly by a corresponding effect of serotonin on entry of sodium ions, would also facilitate the entry of calcium, but it appears that one can occur independently of the other.

Relaxation is assumed to involve a reduction in the intracellular con centration of free calcium ions.

Calcium ions which can be mobilized to activate the contractile apparatus in smooth muscle fibers may originate from two different sources

(Hurwitz and Suria, 1971). One is the pool of calcium that is present in the extra cellular fluid or that is loosely bound to superficial sites in or on the muscle fiber; the other is a tightly bound pool of calcium that is sequestered in some intracelluar location or locations in the fiber (Hurwitz, 1975; Greenberg, et al, 1973)..

Burnstock (1970) has proposed a model of the autonomic neuromuscu lar junction. The essential features are that the terminal portions of the autonomic nerve fibers are varicose, transmitter being released

'en passage1 from varicosities during conduction of an impulse. The effector is a muscle bundle rather than a single smooth muscle cell.

Individual muscle cells are connected by low resistance pathways which allow electrotonic spread of activity within the effector bundle. The 28 sites of these low resistance pathways appear to be 'gap junctions'

(or nexuses) that vary in size between punctate junctions and functional areas more than 1 ym in diameter. The muscle cells that are directly O innervated, i.e., in close (200 - 1200 A) apposition with the nerve varicosites are directly affected by transmitter released. 'Coupled

cells' adjoin 'directly-innervated cells' via low resistance pathways so that excitatory junction potentials can be recorded. When the muscle cells in an area of an effector bundle become depolarized, an all or none action potential is initiated which propagates through the tissue. Thus,

in some tissues, many cells (termed 'indirectly-coupled cells') are neither directly-innervated nor coupled and yet respond to stimulation of the nerves supplying the organ.

The minimum separation of nerve and muscle varies considerably in

O different tissues. In general it is closest (150 - 200 A) in densely

innervated tissues such as the iris and vas deferens, but may be as wide O as 10,000 A in some large elastic arteries (Burnstock, 1975a). In large

elastic arteries, the adrenergic innervation appears to be diminished, whereas in arterioles and in small arteries the separation of nerve and muscle is minimal and the innervation is greater (Bevan and Su, 1974).

The most important motor components in autonomic control of the

vasculature are the sympathetic nerves (Burnstock, 1975^). The adrener

gic nerve fibers that supply most vessels in the body have their origin

in the pre- or para- vertebral ganglia of the sympathetic nervous system,

but it is possible that some blood vessels in the brain may be innervated

by nerve fibers originating in central catecholamine neurons (Edvinsson, et at, 1973). 29

Serotonin and the Central Nervous System

The central neuronal concentration of serotonin in the brain stem has been shown to affect many distant sites. The cellular groups present

in the midsagittal plane of the brain stem, usually referred to as the nucleus or nuclei of the raphe, have been recognized since the early

days of microscopical neuroanatomy. The term raphe refers to a line,

seam, ridge or any other clearly marked structural feature which is

present in the midsagittal plane in the body (Taber, et al, 1960). The nuclear masses situated along the midsagittal plane in the brain stem

(medulla oblongata, pons and mesencephalon) are at many levels separated

on either side from other cellular aggregations by fiber masses. These

fiber masses permitted, Taber, et al, (1960) and Brodal, et al, (1960 a,

b) in a series of papers, to describe and delineate those groups which

are now referred to as the nuclei of the raphe. Eight different nuclei

were distinguished. In a caudorostral sequence, they are: nucleus raphe

obscurus, nucleus raphe pallidus, nucleus raphe magnus, nucleus raphe

pontis, nucleus raphe dorsalis, nucleus centralis superior, nucleus

linearis intermedius, and nucleus linearis rostralis. By observing

retrogrode cellular changes and using the method of silver impregnation,

Brodal, et al (1960a) described the efferent connections of these nuclei

as consisting of long ascending fibers coming from all the nuclear groups

of the raphe. The afferent connections (Brodal, et al, 1960b) consisted

of fibers from the spinal cord and cerebral cortex ending chiefly in the

nucleus raphe magnus. In 1964, Dahlstrom and Fuxe demonstrated the existence of monoamines in the cell bodies of the brain stem neurons. They described the nerve cells of the catecholamine type extending from the medulla oblongata to the diencephalon as group to group A^>. Similarly the nerve cells of the serotonin type were described extending from the medulla oblongata to the mesencephalon and labeled as group to group

B9 . An important observation was that the localization of the serotonin nerve cells differed from that of the catecholamine cells. Serotonin was found to be present almost exclusively in the various raphe nuclei, while the catecholamine cells occupied a more lateral position caud- orostrally. Serotonin present in the raphe groups (Bi - B9 ) showed extensive projections rostrally toward cortex, caudally toward spinal cord, and throughout the brain stem.

The question of whether serotonin is the only indolealkylamine present in the raphe nuclei is still under investigation. Bjorklund, et al, (1971) have found that some raphe neurons may contain an inddealkylamine other than serotonin. However, Jonsson, et al, (1974) found that serotonin is the predominant indolealkylamine present in raphe neurons. This is consistent with the fact that no other indolealkylamine but serotonin has as yet been detected in the raphe nuclei by biochemical methods (Aghajanian, et al, 1973), although small amounts of the serotonin derivative 5-methoxytryptamine have been found in the hypothalamus

(Green, et al, 1973).

Subsequent mapping of brain neuronal pathways has been attempted by a variety of experimental approaches. For the biogenic amines 31 norepinephrine, dopamine, and serotonin, organization of the specific pathways has been largely demonstrated by the histofluorescence method of Falck, Hillarp, and Carlsson (1962). This method depends on the characteristic fluorescence of specific neuronal elements following exposure of the tissue to paraformaldehyde vapor.

Histologically, certain features of the raphe nuclei indicate that they may constitute not only an anatomical but also a functional entity.

It has been suggested that they are part of the morphological substratum of the ascending activating system of the brain stem (Taber, et al, 1960).

Ohta (1975) has suggested that an alpha adrenergic mechanism may be involved in the cerebral cortex and brain stem. Recently, Scheibel, et alt (1975) demonstrated a neurovascular relationship between the raphe nuclei and the surface of the raphe vessels which ascend from the basilar artery. It was proposed that the raphe nuclei may play a role in:

(1 ) neurosecretion, releasing some active agent into the underlying vascular system, (2 ) responding to specific circulating substances, i.e., a chemosensor function, and (3) responding to changes in tone and/or diameter of the vessel wall, i.e., a mechanoreceptor function. Lai, et alt (1975) has shown that the microvessels of rat brain contain norepinephrine and the enzymes involved in its synthesis and degradation.

Thus the relationship between the neurovascular and the central nervous system may contain some kind of regulatory function.

Serotonin has been implicated as a neurotransmitter in the media tion of pain and in the mechanism of migraine. 32

Pain, the main symptom of migraine headache, is considered to be peripheral in origin because it is provoked by mechanical (dilatation- pulsation of the extracranial vessels) and/or biochemical (direct action on vascular and paravascular nociceptors due to chemical substances) stimulation (Sicuteri, et al, 1974). Akil and Liebeskind (1975) suggests that serotonin has an important role in central pain modulation. It is interesting that serotonin levels of the plasma fall at the onset of a migraine attack (Anthony and Lance, 1972). Serotonin is also a potent vasoconstrictor of cerebral and carotid vessels (Lowe and Gilboe, 1973), and an increase in the major metabolite of serotonin, 5 -hydroxyindoleacetic acid, has been inconsistently demonstrated during a migraine attack (Dalessio, 1974). It has been suggested that the preheadache vasoconstrictor phenomena represents neurogenic vasospasm of the innervated system at the base of the brain and the pial arteries

(Dalessio, 1974). Nielsen and Owman (1967), by fluorescence histochem istry, showed that most of the pial arteries at the base of the brain are supplied by adrenergic nerves. Vessels emanating from the carotid system have a more pronounced innervation than those belonging to the vertebral system. However, sympathetic nerves have been questioned as to their role, if any, in the regulation of the vascular tone in large canine cerebral arteries (Toda and Fujita, 1973). Vessels supplying the cerebral vasculature and the cerebral vessels themselves show responses to serotonin which may differ from the rest of the organism.

Vidrio and Hong (1975) observed that serotonin, in the dog, produces vasoconstriction in the internal and vasodilatation in the 33 external carotid vascular beds. A difference in the mechanism of action of ergotamine has been suggested between canine arterial vascular smooth muscle and canine femoral and saphenous veins (Miiller-Schweinitzer, 1976).

Toda and Fujita (1973) observed a change in sensitivity to nore pinephrine and serotonin between the canine mesenteric and cerebral arteries. It appears that the blood vessels, approaching, entering and those that are part of, the cerebral vasculature differ in their contractile responses to the biogenic amines.

The vessels chosen for this study were the basilar, middle cerebral, and posterior cerebellar arteries. Branches of the basilar and posterior cerebellar supply areas rich in serotonin and norepine phrine respectively.

Problem of Thesis

Controversy exists as to the nature of the serotonergic receptor.

Alpha adrenergic antagonists have the ability to block non-adrenergic receptors. Cross reactivity exists between classical and adrenergic agonists and antagonists and the serotonergic receptor.

The following study was designed to elucidate the nature of the serotonergic receptor in vascular smooth muscle. The evidence presented supports the concept that serotonin elicits constriction of vascular smooth muscle by stimulation of alpha adrenoceptors on the muscle cell membrane. This alpha receptor differs from that of the 34 classical alpha adrenoceptor. Evidence is presented for the existence of two subclasses of the alpha adrenergic receptor which we now designate alphai and aZpfag. METHODS AND MATERIALS

Studies on Rat Smooth Muscle

Male Sprague-Dawley rats (Laboratory Supply Company, Indianapolis,

Indiana) weighing 350-500 grams, were killed by cervical dislocation.

Thoracic aortae and small mesenteric arteries (500 micron, O.D.) were removed, placed into physiologic saline solution (PSS) at pH 7.4, 22-24°C and oxygenated with 95% 0£ - 5% CO2 . The blood vessels were cleaned of adhering fat and fascia and cut into helical strips under a dissecting microscope. The vascular strips were then suspended in a muscle bath between a stationary pyrex rod and a Grass FT 03C force displacement transducer. The PSS in the muscle bath was slowly warmed to 37.5 ±

0.19°C with a water circulator constant temperature pump. The slow warming of the vascular strips to 37.5°C was found to enhance the stability of the rat vessels and provide reproducible responses to the agonists throughout the entire experimental period. The arterial strips were al lowed to equilibrate to 37.5°C for three hours prior to challenge with any agonist. The PSS in the muscle bath was replaced every twenty minutes throughout the equilibration period. Force development in response to vasoactive agents was recorded on a Beckman Type R dynograph recorder and

Grass Model 5 polygraph. The PSS contained (mM) : sodium chloride, 125; potassium chloride, 4.7; magnesium chloride, 1.2; calcium chloride,

35 1.6; sodium bicarbonate, 24.8; monosodium dihydrogen phosphate, 1.18; dextrose, 10; sucrose, 50; and calcium disodium ethylenediaminetetra- acetate, 0.026.

Effect of Serotonin and Norepinephrine on Rodent

Vascular Smooth Muscle

Each vascular strip was stretched to the optimal portion of its passive length-active tension curve with the method described by

Greenberg, et al, 1975. A passive force of 25G±25 milligrams was placed on mesenteric artery strips (1 mm width X 7 mm in length) whereas a passive force of 497 ± 23 milligrams was placed on thoracic aortae

(1-2 mm width X 7-11 mm in length). After a three hour equilibration period, complete cumulative concentration effect curves were constructed for serotonin or norepinephrine employing the technique of Van Rossum

(1962). Responses to a concentration of agonist were obtained. After the response had achieved equilibrium, the next higher concentration of agonist was added to the muscle bath. This procedure was repeated until successive additions of a higher concentration of agonist produced no further increment in force. The agonist was then washed out of the muscle bath until basal force was reestablished. Thirty minutes after return of force to pre-agonist value, the responses to norepinephrine or serotonin were re-determined. This procedure was repeated over the time period employed in the studies with the adrenergic receptor blocking agents (see below). Similar studies were performed employing potassium chloride as a non-receptor mediated stimulant of vascular smooth muscle. 37

Each vascular strip was exposed to only one agonist. The contractile responses of each vascular strip to norepinephrine or serotonin was

expressed as a percentage of the maximum response of the vascular strip

to the first concentration effect curve constructed. The concentration

of agonist necessary to elicit 50 percent of the maximum contractile response of the muscle to that agonist was determined by the method of

Schild (1959).

Effect of Autonomic Blocking Agents on the Contractile

Responses of Vascular Smooth Muscle to Serotonin,

Norepinephrine and Potassium Chloride

This series of experiments was designed to evaluate the qualitative

effects of serotonergic, alpha adrenergic and beta adrenergic receptor

antagonists on the contractile responses of rat thoracic aortae and

mesenteric arteries to serotonin and norepinephrine. Rat vascular

smooth muscle, prepared for recording as described above, were chal

lenged with either norepinephrine and potassium chloride or serotonin

and potassium chloride prior to and during incubation with methysergide,

phentolamine or propranolol. Each antagonist was in contact with the

muscle preparations for thirty minutes prior to and throughout re-

evaluation of the concentration effect curves for the agonists. 38

Effect of Phentolamine, Methysergide and Tolazoline on

Serotonin and Norepinephrine Induced Contractions of Rat

Vascular Smooth Muscle

Helical strips of rat thoracic aortae and mesenteric arteries were prepared for the isometric recording of force development as described above. Complete cumulative concentration response curves were obtained for either norepinephrine or serotonin. Twenty minutes after the res toration of force to control values, upon washout of the agonist, either phentolamine, tolazoline or methysergide were added to the muscle baths containing the vascular strips. The antagonists were allowed to equili brate with the helical strips of blood vessels for thirty minutes. The responses to the agonist were then re-evaluated and the agonist and antagonist were washed out of the muscle bath. Twenty minutes after restoration of force to basal values the next higher concentration of antagonist was added to the muscle bath. Thirty minutes later the responses to the agonist were then re-evaluated. This procedure was repeated until maximal blockade of the agonist or 1 X 10-^M antagonist was achieved. The concentration of antagonist necessary to increase the ED^q of the agonist by a factor of 2 was calculated by the method of Arunlakshana and Schild (1959).

Briefly, the concentration of agonist necessary to produce fifty percent of maximum response was obtained for each vascular strip from a graph of the responses between twenty to eighty percent of maximum response versus the logarithmic concentration of antagonist. The 39 logarithm of the ratio of concentration of agonist in the presence and absence of antagonist minus 1 was plotted as a function of the logarithm of the concentration of antagonist. The best fitting line was drawn through the points until the line crossed the abscissa. This point represents the concentration of antagonist necessary to increase the

ED5 0 of the agonist by a factor of two. The log (dose ratio minus 1) equals to zero (the abscissa of the graph, PA2 ) when the concentration of agonist in the presence and absence of antagonist equals two, since

Log (2 -1 ) = Log C D = 0 .

In a separate series of experiments, the pA£ values of the antagonists against norepinephrine and serotonin were evaluated in thoracic aortae and mesenteric arteries incubated with cocaine, 5 X 10”^M to inhibit neuronal reuptake of the agonists. Vascular strips, prepared as described above, were incubated with cocaine for three hours prior to and throughout evaluation of the inhibitory effect of the receptor antagonists on the contractile responses to norepinephrine and serotonin.

ED5 0 and pA2 values were determined as described above.

Effect of Catecholamine Depletion and Blockade of

Adrenergic Neuronal Reuptake on the Contractile

Responses to Serotonin and Norepinephrine

The rat thoracic aortae is sparsely innervated with adrenergic nerve terminals whereas the rat mesenteric artery is densely innervated with adrenergic nerves and varicosities (Burnstock, et al, 1972).

Despite this fact both aortae and mesenteric arteries can synthesize and 40 store norepinephrine and serotonin (Berkowitz, et at, 1972; Jarrott, et at, 1975). Furthermore, these vessels appear to degrade both agonists (Jarrott, 1975). Since serotonin is a substrate for mono amine oxidase (type A), it could alter the metabolism of norepinephrine and produce an increase in norepinephrine at the receptor site without directly stimulating the release of norepinephrine. Therefore, a series of experiments were performed on vascular strips obtained from reserpine- treated rats and on vascular strips incubated with cocaine (5 X 10“®M) to inhibit neuronal reuptake of norepinephrine and serotonin. Some experiments were also performed on vascular strips obtained from reserpine treated animals and subsequently incubated with cocaine

(5 X 10~^M) to prevent repletion of the nerve terminals with either norepinephrine or serotonin (Greenberg, et at, 1974; Snipes, et at, 1967).

Male Sprague Dawley rats (350-500 grams) were pretreated with reserpine hydrochloride (2.5 mg/kg/day i.p.) for six consecutive days. Vascular strips were then removed and prepared for recording as described above.

After a three hour equilibration period, cumulative concentration effect curves were constructed for serotonin or norepinephrine. The concentra tion response curves were repeated in the presence or absence of increasing concentrations of phentolamine, tolazoline or methysergide.

Each vascular strip was exposed to one agonist and one antagonist. ED5 0 and pA£ values were calculated as described above.

In a separate series of experiments vascular strips, prepared as described above, were incubated and equilibrated in FSS containing cocaine hydrochloride (5 X 10“®M). After the three hour equilibration 41 period complete concentration effect curves were constructed for serotonin and norepinephrine in the presence and absence of tolazoline, phentolamine or methysergide. These experiments were repeated on vascular strips obtained from animals pretreated with reserpine

(2.5 mg/kg/day for six consecutive days). ED5 Q and pA£ values were calculated as described above.

Effect of Tolazoline on Phentolamine Induced

Blockade of the Respones to Serotonin

Rat thoracic aortae and mesenteric arteries were prepared for recording as described above. Tolazoline (1 X 10” ^ or 1 X 10“^M) was present in the muscle bath and PSS throughout the equilibration period and during the entire experiment. Complete cumulative concentration effect curves were constructed for serotonin and norepinephrine in the presence and absence of phentolamine or methysergide. ED5 Q values and the maximal increase in force produced by the agonist were recorded.

Effect of Calcium Ion on the Adrenergic Blocking

Activity of Phentolamine

From the data obtained in the following experiments, it was apparent that phentolamine was ten times more potent an antagonist of norepinephrine than had been reported previously by other investigators (for references see Furchgott, 1972). While several factors may account for the dis crepancy between the author's data and that of other investigators, it was qjparent that the calcium ion concentration used in the PSS was significantly lower than that reported in most studies (1.6 vs 2.0-2.5mM). The effect of an increased calcium ion concentration on the adrenergic receptor blocking activity of phentolamine was evaluated. Helical strips of rat thoracic aortae were prepared for recording as described above except that the PSS contained 2.5mM calcium chloride. Complete cumula tive concentration effect curves for norepinephrine were constructed in the presence of increasing concentrations of phentolamine. ED5 0 and pA£ values were calculated by the method of Arunlakshana and Schild (1959) as described above.

Binding of 2-C^-Serotonin to Rat Thoracic Aortae

Rats were killed by cervical dislocation and their thoracic aortae removed, cleaned of adhering connective tissue, cut into helical strips and incubated in oxygenated PSS for four hours at 37.5°C. The vascular strips ( 1 mm in width by 1 0 mm in length) were then incubated for variable time periods in PSS containing 2-Cl^-serotonin (New England

Nuclear, Boston, Mass.; 25yCi/10"^M 5-HT). Similarly, vascular strips were incubated for thirty minutes in the presence and absence of either

cocaine, 5 X 10”^M; tolazoline, 1 X lCT^M; phentolamine, 1 X 10“®-1 X

10“5m; or norepinephrine, 1 X 10“^-1 X 10-"*M; alone or in combination.

The vascular strips were then incubated for ten minutes with 2-C-^-

serotonin (25yCi/10”^M) in the presence and absence of drugs. The vascular strips were then removed, blotted on Whatman No. 5 filter paper

and digested overnight in 6 N NaOH. The excess alkali was neutralized with 6N HC1, diluted with Aquasol-2 (New England Nuclear, Boston, Mass.) A3

and allowed to stand In the cold-room overnight. The samples were

counted in a Packard TriCarb liquid scintillation counter for ten minutes. All samples were corrected for quenching with an external

standard, and for the counting efficiency of the counter. Data were

expressed as disintegrations per minute per milligram of aortae.

Studies on Canine Venous Smooth Muscle

Mongr.el dogs (15-25 kg) of either sex were anesthetized with pento

barbital (35 mg/kg i.v.). Saphenous veins were excised, placed in

physiologic saline solution (PSS) at room temperature, cleaned of

adhering connective tissue and fat and cut into helical strips approxi

mately 1 mm in width by 7 mm in length. The muscle strips were placed

in a 10 ml muscle chamber filled with oxygenated PSS at room tempera

ture and were mounted between a stationary pyrex rod and Grass FT 03C

force displacement transducer for isometric recording of force develop

ment. The heater of the circulator bath was turned on and the muscles

slowly allowed to equilibrate to 37.5°C. The PSS in the muscle bath was

replaced every twenty minutes with fresh oxygenated PSS. Saphenous vein

strips were allowed to equilibrate for three hours prior to experimenta

tion. The PSS maintained at 37.5°C and aerated with 95% 0£ - 5% CC>2

(pH 7.A) was of the following composition (mM): sodium chloride (125);

potassium chloride (A.7); magnesium chloride (1.2); calcium chloride

(1.6); sodium bicarbonate (2A.8); monosodium dihydrogen phosphate (1.18);

dextrose (10); sucrose (50) and calcium disodium ethylenediaminetetra-

acetic acid (0.026). 44

Contractility Studies

Saphenous veins were placed at the optimal portion of their passive length active-tension curve as described previously (Greenberg, et at,

1974). Concentration effect curves of the saphenous veins were deter mined to cumulative additions of norepinephrine, serotonin or potassium ion to the muscle baths. Each vein strip was exposed to only one agonist. Upon removal of the agonists from the muscle bath and return of force to baseline values, the responses to each of the agonists were then re-determined in the presence or absence of phentolamine (1 X 10"® -

1 X 1-“% ) , tolazoline (1 X 10"® - 1 X 10“% ) , or methysergide (1 X

10“® - 1 X 10"%), alone or in combination. Each concentration of antagonist was allowed to remain in contact with the saphenous veins for thirty minutes before, as well as throughout, re-evaluation of the responses to the agonist. These experiments were repeated in a second series of experiments on helical strips of canine saphenous veins incubated with 2 X 1 0 " % cocaine hydrochloride during the equilibration period and through the entire experiment.

The contractile responses of canine saphenous vein to each agonist before and during administration of the adrenergic receptor blocking agents were expressed as a percent of the maximum response to each agonist in the absence of the blocking agent. The concentration of each agonist necessary to elicit fifty percent of the maximum response of the muscle to that agonist (ED5 Q) was calculated by the method of Schild

(1959). The concentration of antagonist necessary to increase the ED5 Q 45

of each agonist by a factor of two was calculated by the method of

Arunlakshana and Schild (1959) with the experimental criteria estab

lished by Furchgott (1972). Briefly, these criteria include adequate

time for equilibrium of antagonist with the muscle (thirty minutes),

attainment of equilibrium responses to the agonist, and the presence

of adequate time controls.

Effect of Serotonin on 7-^H-Norepinephrine Efflux

Helical strips of canine saphenous vein were prepared for measure

ment of 7-%-norepinephrine efflux as described by Muldoon, et al, (1976).

Briefly, helical strips of veins were incubated at 37.5°C for four

hours in Krebs-Ringer solution containing 7-%-norepinephrine at 3 X

10~7m (specific activity 8 . 8 Ci/millimole, Amersham/Saerle Corporation

Des Plaines, Illinois). The strips were then rinsed in fresh Krebs-

Ringer solution and mounted for superfusion. The muscle strips were

suspended in a moist, tunnel-shapped chamber maintained at 37°C; the

strips were superfused with oxygenated Krebs-Ringer solution (37°C;

pH 7.4) at 5 ml/min with a constant flow roller pump. The veins were

then connected to a Grass FT 03C strain guage for the isometric

recording of force development. Each vein was stretched with a passive

force of 3 grams. After stress relaxation the resting force was stable

throughout the remainder of the experiment. After a thirty minute

equilibration period, the superfusate was collected at two minute

intervals for direct evaluation of the total radioactivity in the super

fusate. After a fourteen to sixteen minute control period, the muscle

strips were superfused with Krebs solution containing 1 X 10“7m serotonin 46 until a steady state contractile response was obtained. At the peak of the contractile response to serotonin, phentolamine (1 X 10~^M) was added to the superfusate. Sixteen and thirty-two minutes after the infusion of phentolamine and serotonin, respectively, the infusion was terminated and the muscle strips allowed to return to resting force for fourteen to sixteen minutes prior to termination of the experiment. At this time the muscle strips were blotted and weighed. In some experi ments, cocaine (1 X 10~^M) was present in the superfusate throughout the entire experiment to inhibit the active reuptake of norepinephrine and serotonin by the adrenergic nerves

Viability of Norepinephrine Release

Two platinum wires (0.5 mm diameter, 10 cm in length) were placed parallel to the veins in the tissue chamber. Both the electrodes and veins were continuously superfused. At the end of each experiment, the veins were electrically stimulated with square wave pulses (8 volts; 2 msec duration; 2 to 5 Hz) provided by a direct current power supply and a switching transistor (RCA 2H-3034) triggered by a Grass S- 8 8 stimulator. The stimulus was applied for five minutes. All prepara tions tested demonstrated an increase in the efflux of 7 - % - norepine phrine. 47

Radioactivity Measurements

One ml aliquots of the superfusate, and the eluates and effluents from the Dowex 50 and alumina columns were added to 10 ml of Insta-Gel

(Packard Instrument Company, Inc., Downers Grove, Illinois). The radio activity was measured with a Packard Tri Carb liquid scintillation counter. Corrections for quenching were performed with an external standard. The counting efficiency was 44 percent. Samples were counted for ten minutes. Samples containing the extracted and isolated nor epinephrine were counted for a sufficient time to reach 1 0 , 0 0 0 counts.

Recovery of 7-3h norepinephrine was 99.6 ± 1.0%. The results are expressed as disintegrations per minute per minute of superfusion for the efflux experiments and disintegrations per minute per collection period for the distribution of 7 - % norepinephrine.

Statistical Analysis of Data

For each experiment, the number of vascular strips represents the number of animals used as well. The data are expressed as means ± the standard error of the means. Data were analyzed with analysis of variance and Duncans New Multiple Range Test and Students t-test for paired and unpaired data (Steel and Torries, 1960). pA£ and ED5 Q values were calculated by the method of Arunlakshana and Schild (1959). A

P value of 0.05 or less was chosen for statistical significance of the difference between the means. 48

Drugs Used

L-norepinephrine bitartrate (Levophed, Sterling Winthrop Research

Institute, Rensselear, New York), phentolamine hydrochloride, tolazoline hydrochloride, reserpine hydrochloride (Ciba Pharmaceutical Company,

Summit, New Jersey), cocaine hydrochloride (Merck Chemical Company,

Rahway, New Jersey), serotonin creatinine sulfate (Sigma Chemical

Company, St. Louis, Missouri), serotonin binoxalate (The Ohio State

University Hospitals, Columbus, Ohio), methysergide maleate (gift from

Dr. T. F. Burks, University of Texas Medical School, Houston, Texas), propranolol hydrochloride (Ayerst Laboratories, Montreal, Canada),

2-C^-serotonin binoxalate (New England Nuclear, Boston, Massachusetts). RESULTS

The contractile responses of rat thoracic aortae and mesenteric arteries to norepinephrine and serotonin are summarized in Figure 3.

The rat thoracic aorta is almost entirely devoid of adrenergic innerva tion (Burnstock, et al> 1972). In this preparation norepinephrine is a more potent constrictor of smooth muscle than is serotonin. The slopes of the linear portion of the concentration effect curves do not differ significantly (P>0.05) between the two agonists (Figure 3, left panel).

Norepinephrine was approximately 160 times more potent a constrictor of rat thoracic aortae than was serotonin. In contrast to these findings, the sensitivity of rat mesenteric arteries to norepinephrine and serotonin was similar. Norepinephrine was 2.8 times more potent a constrictor agent than was serotonin. The slope of the linear portion of the concentration effect curve significantly deviated from parallelism

(P<0.05). Furthermore, the sensitivity of the rat mesenteric artery to serotonin was greater than that of the aorta, whereas the sensitivity to norepinephrine was diminished (Figure 3, right panel). Thus, serotonin and norepinephrine both elicit contraction of rat thoracic aortae and mesenteric arteries. The sensitivity of both preparations to norepine phrine is greater than that to serotonin. Finally, the thoracic aortae is more sensitive to norepinephrine and less sensitive to serotonin than is the rat mesenteric artery. This may reflect the presence of adrenergic

49 » 50

FIGURE 3

COMPARISON OF VASCULAR SMOOTH MUSCLE RESPONSES TO 5-HT AND NE

Comparison of vascular smooth muscle responses to serotonin (5-HT) and norepinephrine (NE). The ordinate represents the contractile responses of the muscle (expressed as a percent of maximum contractile response) to norepinephrine (closed circles) and serotonin (open circles).

The abscissa represents the concentration of agonist. Each value represents the mean and standard error of the mean. N = the number of

rats used to supply the vascular strips. The concentration of agonist necessary to elicit fifty percent of maximum response (ED5 0 ) for norepinephrine and serotonin differ significantly from each other

(P<0.05) within the aorta and the mesenteric artery. CONTRACTILE FORCE (%) t SEM 0 0 1 0 4 80 0 2 60 OCNRTO O AOIT OCNRTO O AGONIST OF CONCENTRATION AGONIST OF CONCENTRATION 0" O 0 09 0' I" I' I 5 0n O 0 09 08 07 0 I0"5 6 I0‘ I0-7 I0'8 10 IO‘ I0'9 I0"n '5 I0 I0'6 I0"7 '8 I0 IO‘ 10 I0'9 10"" HRCC OT MSNEI ARTERY MESENTERIC AORTA THORACIC OPRSN F ACLR MOH UCE RESPONSES MUSCLE SMOOTH VASCULAR OF COMPARISON O HT AD NE AND T -H 5 TO • —o HT -H 5 o o— ------NE ED50=5.53+0.64xI0-8 =5 V N=35 /

52 nerves within the latter preparation modifying the disposition of these vasoconstrictor substances within the vascular smooth muscle (Berkowitz, et aty 1972; Jarrott, et al, 1975).

Effect of Autonomic Blocking Agents on the Contractile

Responses of Rat Arterial Smooth Muscle to Serotonin and Norepinephrine

The effect of a thirty minute incubation of rat thoracic aortae and mesenteric arteries with the alpha adrenergic receptor blocking agent phentolamine, the serotonergic receptor blocking agent methyser gide, the beta receptor blocking agent propranolol, or with the drug free PSS on the contractile responses to norepinephrine and serotonin are summarized in Figures 4 and 5. The contractile responses of rat thoracic aortae to both norepinephrine and serotonin were stable throughout the entire experimental procedure (Figure 4, upper left panel).

However, phentolamine inhibited the contractile responses of this smooth muscle preparation to both norepinephrine and serotonin. Similar findings were obtained when methysergide, a potent serotonergic receptor blocking agent, was employed as the autonomic antagonist (Figure 4, upper right and lower left panels). The possibility that this inhibitory effect was shared by high concentrations of any adrenoceptor antagonist was eliminated when propranolol failed to significantly inhibit the contractile responses to either norepinephrine or serotonin (Figure 4, lower left panel). Similar findings were observed in the densely innervated mesenteric artery (Figure 5). Thus these data suggested that 53

FIGURE 4

EFFECT OF RECEPTOR BLOCKING AGENTS ON AORTIC SMOOTH MUSCLE RESPONSES TO 5-HT AND NE

Effect of autonomic receptor antagonists on the contractile

responses of rat thoracic aortae to norepinephrine and serotonin. The ordinate represents the contractile response of the blood vessel

(expressed as a percentage of the maximal control response) to the agonists. The abscissa is the molar concentration of agonist. The

open circles represent the control responses to the agonists. The

closed circles represent the responses to the agonists thirty minutes

after restoration of the basal force of the muscle to control values

upon washout of the agonist from the muscle bath (upper left panel); and

thirty minutes after incubation with phentolamine (upper right panel),

methysergide (lower left panel) and propranolol (lower right panel).

Vertical lines represent the standard errors of the means. Each mean

± S.E.M. represents the responses obtained from three to seven vascular

strips, each vascular strip representing one rat. DEVELOPED FORCE (% MAXIMUM)! SEM IOO- lOOi - 0 5 5 2 5 7 - 5 7 A 25-i 0- 5 C N = 7 0' I* I 8 07 0' to*5 '6 I0 I0"7 '8 I0 IO*9 10*'° N=3 CONTROL CONTROL CONTROL I E TIM MSG \x\0*My\ N OTC MOH UCE SOSS O HT ANDNE N D N A T -H 5 TO ESPONSES R MUSCLE SMOOTH AORTIC ON ft, FET F EETR LCIG AGENTS BLOCKING RECEPTOR OF EFFECT ne OA CNETAIN F AGONIST OF CONCENTRATION MOLAR NE

5HT '5-H HT -H 5 100-1 75- 50- 25- B N=7 Qt 1- I* I* 1* 10*5 10*6 IO*7 IO*8 10-9 IQ-to r 10 PORNLL xO M 5 lxlO‘ •PROPRANOLOL ^CONTROL PHENTOLAMIN CONTROL to *9 io i ------

'8 io NE 1 ------'7 io - T 5-H NE

r '6 io 54 HT -H 5 -5 55

FIGURE 5

EFFECT OF RECEPTOR BLOCKING AGENTS ON MESENTERIC SMOOTH MUSCLE RESPONSES TO 5-HT AND NE

Effect of autonomic receptor antagonists on the contractile responses of rat mesenteric arteries to norepinephrine and serotonin.

The ordinate represents the contractile response of the blood vessel

(expressed as a percentage of the maximal control response) to the agonists. The abscissa is the molar concentration of agonist. The open circles represent the control responses to the agonists. The closed circles represent the responses to the agonists thirty minutes after restoration of the basal force of the muscle to control values upon wash out of the agonist from the muscle bath (upper left panel); and thirty minutes after incubation with phentolamine (upper right panel); methysergide (lower left panel) and propranolol (lower right panel).

Vertical lines represent the standard errors of the means. Each mean ±

S.E.M. represents the responses obtained from three to seven vascular

strips, each vascular strip representing one rat. DEVELOPED FORCE (% MAXIMUM)! SEM lOO-i lOO-i 50- 75- 25 25- 50- 75- A C N EETRC MOH UCE EPNE T 5- N NE AND T -H 5 TO RESPONSES MUSCLE SMOOTH MESENTERIC ON ,-10 10 N =6 TIME CONTROL CONTROL S I MSG 1-9 ■9 x IO'5M

NE. 8 - 1 FET F EETR LCIG AGENTS BLOCKING RECEPTOR OF EFFECT 8 OA CNETAIN F AGONIST OF CONCENTRATION MOLAR 1-7 •7 5-HT HT, T -H 5 5-HT. >-6 6 e n NE 5 lOO-i lOO-i 50- 25- 75- 50- 25- 75- B D ,-10 ,-10 N = 7 CONTROL PHENTOLAMINE CONTROL PROPRANOLOL NE 1*9 I I x x 5-HT, IO‘5M O5M IO'5 1-8 1-8

1-7 ■7•9 1-6 NE 6 - T 5-H - T 5-H NE 56 1-5 •5 the Cl) concentrations of adrenoceptor blocking agents employed non- specifically depressed the contractile integrity of the muscle; (2 ) serotonin acted to release norepinephrine the action of which was sub sequently inhibited by phentolamine, the most specific alpha adrenoceptor

antagonist known (Furchgott, 1972); or (3) serotonin elicits its

contractile responses on rat thoracic aortae and mesenteric arteries by

stimulation of alpha adrenergic receptors located in or on the smooth

muscle cell membrane. These postulates were tested.

Effect of Autonomic Receptor Antagonists on the

Contractile Responses of Rat Vascular Smooth Muscle

to Potassium Chloride

The contractile responses of rat aortic and mesenteric arterial strips

to potassium chloride were reproducible throughout the entire experimen

tal procedure. Neither phentolamine, methysergide, propranolol nor

tolazoline, another adrenoceptor antagonist (see below) inhibited the

contractile responses to potassium chloride (Table 1). Similarly, the

contractile responses of rat thoracic aortae and mesenteric arteries were neither potentiated nor inhibited by inhibition of adrenergic

neuronal re-uptake blockade with cocaine (5 X 10“% ) . Thus these results

suggest that the inhibitory effect of adrenoceptor and "serotonergic

receptor" blockade by phentolamine, tolazoline and methysergide, respec

tively, cannot be secondary to non-specific depression of the vascular

smooth muscle contractile machinery (Table 1). TABLE 1

'EFFECT OF AUTONOMIC BLOCKING AGENTS ON THE CONTRACTILE RESPONSES OF RAT VASCULAR SMOOTH MUSCLE TO POTASSIUM CHLORIDE TABLE 1. EFFECT OF AUTONOMIC BLOCKING AGENTS ON THE CONTRACTILE RESPONSES OF RAT VASCULAR SMOOTH MUSCLE TO POTASSIUM CHLORIDE

CONTRACTILE RESPONSES TO 50 mM KCL (A mg ± S.E.M.)

THORACIC AORTA MESENTERIC ARTERY

INTERVENTION CONCENTRATION Na CONTROL TREATED Na CONTROL TREATED

Timeb — 8 405 ± 60 470 + 80 8 110 ± 29 111 ± 27

Cocaine HClc 5 X 10-6M 1 1 210 ± 30 255 + 2 0 1 1 127 ± 19 133 ± 26

Phentolamine HC1C 1 X 10"5M 8 365 ± 55 375 + 45 8 141 ± 22 137 ± 24

Methysergide0 1 X 10-5M 8 470 ± 105 605 + 105* 8 143 ± 29 200 ± 40*

Tolazoline0 1 X 10"4M 8 340 ± 65 500 + 45* 8 137 ± 22 188 ± 13*

Propranolol0 1 X 10"5M 8 215 ± 60 215 + 50 8 1 2 0 ± 2 2 111 ± 27 a - N, number of experiments. b - Muscle strips were exposed to PSS with 1.60 mM calcium for sixty minutes before the responses to potassium ion were re-evaluated.

c - Muscle strips were exposed to PSS with the antagonist for sixty minutes before the responses to HC1 were re-evaluated.

* - Differs significantly from control responses (P<0.05). 60

Blockade of Adrenergic Neuronal Reuptake and Depletion

of Norepinephrine on the Contractile Responses of Rat

Vascular Smooth Muscle to Serotonin and Norepinephrine

The postulate that serotonin-mediated contraction of rat vascular

smooth muscle was associated with release of norepinephrine from

adrenergic nerves was pharmacologically evaluated. Cocaine (5 X 10"^M) was employed to inhibit neuronal reuptake of norepinephrine. This con

centration of cocaine inhibited the contractile responses to tyramine,

an indirectly acting sympathomimetic amine. Since tyramine must be

taken up into the adrenergic nerve terminal to elicit release of

norepinephrine, this was taken as suggestive evidence of the pharmaco

logic efficacy of cocaine in these preparations (Greenberg, et at, 1974).

The contractile responses of the rat thoracic aortae to serotonin was

significantly potentiated after incubation for thirty minutes with

cocaine (Figure 6 A). In contrast, and as would be expected in a prepara

tion with a sparse adrenergic innervation, the contractile responses of

rat thoracic aortae to norepinephrine demonstrated only a small, but

significant increase in sensitivity (shift of the ED5 Q to the left).

These data are summarized in Figure 6 B. Cocaine-induced potentiation of

the contractile responses of rat mesenteric artery to serotonin and

norepinephrine were potentiated to a similar degree (Figures 6 C and 6 D).

However, the enhancement was manifest as an increase in the threshold of

the concentration effect curve rather than a parallel shift to the left

of the entire curve (.compare Figures 6 A and 6 B with 6 C and 6 D). Higher

concentrations of cocaine did not result in potentiation of the 61

FIGURE 6

EFFECT OF COCAINE ON VASCULAR SMOOTH MUSCLE RESPONSES TO NOREPINEPHRINE AND SEROTONIN

Effect of adrenergic neuronal reuptake blockade by cocaine

(5 X 10_6M) on the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin and norepinephrine. The ordinate represents the change in force (expressed as percentage of maximal con tractile force) of the vascular strips produced by the agonists. The abscissa represents the molar concentration of agonist. The vertical lines are the standard errors of the means. Each mean represents the responses of seven or eleven vascular strips. Each vascular strip was obtained from a single rat. The closed circles are the control responses. The open circles are the responses thirty minutes after the addition of cocaine to the muscle bath. An asterisk denotes that the responses of the smooth muscle to the agonists significantly differ in the presence and absence of cocaine (P<0.05). CONTRACTILE FORCE (%OF CONTROL)! SEM 200-1 160- 120 100 - 0 4 - 0 8 A 40- 60- 80- 20 C 0- 0 - - - - O9 ' I* IO*6 IO*7 I O'8 IO*9 EPNE T OEIEHIE SEROTONIN D N A NOREPINEPHRINE TO RESPONSES HT ( ls ) r e t i l oles/ (m T -H 5 FECT O COCAN O VSUA SOT MUSCLE SMOOTH VASCULAR ON AINE C O C OF T C E FF E 1-9 * P<0.05 * =11N HT ( es/ier NRPNPRN ( es/ier) /lite s le o (m NOREPINEPHRINE r) /lite s le o (m T -H 5 CONTROL COCAINE T 5xlO'6 M 5xlO'6 8 TT

■7 T 1-6 EETRC ARTERY MESENTERIC

HRCC AORTA THORACIC * IO'5 100-1 60- 80- 40- 40- 60- 20 80- 20 D - - OEIEHIE mol lt r) /lite s le o (m NOREPINEPHRINE P<0. 5 .0 0 < *P 01 1* 1- I' I' IO'5 IO'6 IO'7 10-6 10*9 10-1° N=7 P<0.05 * N=7

62 concentration effect curves of the mesenteric arteries to serotonin or norepinephrine. Rather non-specific depression was observed (data not shown). These findings suggest that serotonin may release norepinephrine or alternatively serotonin may be inactivated by a cocaine sensitive process located within the blood vessel. Either of these postulates would result in cocaine-mediated enhancement of the contractile responses to serotonin as well as norepinephrine. If the latter postulate were correct then phentolamine should still inhibit the responses resulting from direct alpha adrenergic stimulation by serotonin. However, phen tolamine may also inhibit a possible serotonin-mediated release of norepinephrine. Were serotonin acting to release norepinephrine from adrenergic nerves within the vascular smooth muscle cell then depletion of norepinephrine stores with pharmacologic doses of reserpine should abolish the contractile responses to serotonin. Furthermore, cocaine

should not potentiate the responses to serotonin in vascular strips obtained from reserpined treated rats. These postulates were tested.

The contractile responses of thoracic aortae and mesenteric arteries

to serotonin were significantly enhanced, rather than inhibited, when

obtained from rats pretreated with reserpine (2.5 mg/kg/day for six

consecutive days) as compared with the responses of the vasculature from normal rats. Reserpine-induced facilitation of the contractile responses

to serotonin and norepinephrine were similar in aortic and mesenteric

arterial strips. However, whereas amine-depletion with reserpine 64 potentiated the contractile responses to serotonin, the responses of the densely innervated mesenteric artery to norepinephrine did not differ from the responses obtained in mesenteric arteries from untreated rats.

These data are summarized in Figures 7 and 8 and Table 2. The contrac tile responses of thoracic aortae and mesenteric arteries obtained from reserpine (2.5 mg/kg/day i.p. for six consecutive days) treated rats to serotonin and norepinephrine were potentiated when cocaine (5 X 10“^M) was added to the muscle baths. The magnitude of the potentiation was similar to that observed in vascular strips obtained from control rats

(Table 2 compare with Figure 6 ). These data suggest that serotonin- mediated contraction of rat vascular smooth muscle is not mediated by release of norepinephrine from adrenergic nerve terminals innervating the vascular smooth muscle cells. Furthermore, the data are consistent with the postulate that serotonin may be inactivated by a cocaine sensi tive process. Inhibition of this process by cocaine may allow for a greater concentration of serotonin to accumulate at receptor sites and thereby elicit an enhanced contractile response. Finally, since reser pine pretreatment did not inhibit the contractile responses to serotonin, phentolamine-mediated inhibition of the contractile responses of rat vascular smooth muscle may be secondary to blockade of an alpha-receptor mediated component of the contractile response to serotonin.

Analyses of the Inhibitory Effect of Phentolamine on the

Contractile Responses to Serotonin

The results of the previous studies suggested that serotonin may elicit contraction of rat vascular smooth muscle by stimulation of the TABLE 2

ed5 0 VALUES OF NOREPINEPHRINE AND SEROTONIN ON RAT THORACIC AORTAE AND MESENTERIC ARTERIES TABLE 2. ED5 0 VALUES OF NOREPINEPHRINE AND SEROTONIN ON RAT THORACIC AORTAE AND MESENTERIC ARTERIES

THORACIC AORTAMESENTERIC ARTERY

Treatment NOREPI SEROTONINNOREPI SEROTONIN

ed5 0 x io_9a ed5 0 X io-7a ed5 0 x io_?a ed5 0 X io_7a

Control 4.07 ± 2.04 6.78 ± 0.26 5.53 ± 0.64 1.92 ± 0.84 (39)b (44) (35) (38)

Cocaine (5 X 10-6 M) 2 . 1 ± 1 .0 2 * 1.1 ± 0.4* 4.91 ± 0.57 0.62 ± 0 .2 1 * (7) (1 1 ) (1 1 ) (1 1 )

Reserpine (2.5 mg/kg/day)c 1.3 ± 0.34* 3.33 ± 1.01* 5.65 ± 1.67 0.79 ± 0.21* (15) (1 1 ) (15) (15)

Reserpine + Cocaine (5 X 10_bM) 0.62 ± 0.24* 0.63 ± 0.31* 4.31 ± 1.92 1 . 0 ± 0 . 2 1 (1 1 ) (1 2 ) (1 2 ) (1 2 )

a - ED5 0 is the concentration of agonist (moles/liter) necessary to elicit fifty percent of maximum resoonse. b - Numbers in parentheses reflect the number of vascular strips employed in the calculation of the mean. c - Reserpine was administered i.p. for six consecutive days.

* - Differs significantly (P<0.05) from control.

O' O' 67

FIGURE 7

EFFECT OF RESERPINE TREATMENT ON VASCULAR RESPONSES TO 5-HT

Effect of reserpine pretreatraent (2.5 mg/kg/day i.p. for six consecutive days) on the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin. The ordinate represents the contractile force (expressed as a percentage of the maximal contractile force) of the vascular strips produced by- serotonin. The abscissa represents the molar concentration of serotonin. The left panel is the thoracic aorta. The right panel is the mesenteric artery. The vertical lines are the standard errors of the means. The numbers in parentheses are the number of arterial strips (each obtained from an individual animal) employed in the calculation of the mean value. The ED^q for serotonin is also displayed. An asterisk denotes that the responses of thoracic aortae and mesenteric artery from normal and reserpine treated rats to serotonin significantly differ (P<0.05) from each other. CONTRACTILE FORCE (PERCENT)t SEM 0 0 1 0 4 60 80- 20 FETO RSRIE RAMN O VSUA RSOSS O - T 5-H TO RESPONSES VASCULAR ON TREATMENT RESERPINE OF EFFECT - Reserpine (2.5mg/Kg/day)(ll)y ED50 = 3.33± I.0 ED50 = 3.33± P<0.05 * HRCC AORTA THORACIC HT oles/liter) m ( T -H 5 I x 0 I‘6 I0‘ 7 I0‘ 07 * I0'7M D067 02 0 7M I0‘ x i0.26 78 ED50=6 oto ( ) 20 4) (4 Control

0 1 -5 lOOn 0 4 80 60- ED5 Reserpine (2.5mg/Kg/day) o 9+.2I + 79 0 = EETRC ARTERY MESENTERIC HT ( oles/liter) (m T -H 5 x O7 V IO_7M (15) D0 .2 .4xl 7 ‘ l0 x 0.84 ED50= 1.921 oto ( ) 8 (3 Control *

i-5 69

FIGURE 8

EFFECT OF RESERPINE PRETREATMENT ON VASCULAR RESPONSES TO NE

Effect of reserpine pretreatment (2.5 mg/kg/day i.p. for six consecutive days) on the contractile responses of rat thoracic aortae and mesenteric arteries to norepinephrine. The ordinate represents the contractile force (expressed as a percentage of the maximal contractile force) of the vascular strips produced by norepinephrine. The abscissa represents the molar concentration of norepinephrine. The left panel is the thoracic aorta. The right panel is the mesenteric artery. The vertical lines are tfre standard errors of the means. The numbers in parentheses are the number of arterial strips (each obtained from an

individual animal) employed in the calculation of the mean value. The

ED5 0 for norepinephrine is also displayed. An asterisk denotes that the responses of thoracic aortae and mesenteric artery from normal and reser pine treated rats to norepinephrine significantly differ (P<0.05) from each other. CONTRACTILE FORCE (%) + SEM 0 0 1 0 2 80- 40- 60- - - i ~ ED50= 1.30 +0.34 01 I" I"0I* 0' I* I‘6 I0"5 6 I0‘ IO"10 IO*7 IO*9 '8 I0*12 I0"n I0 Reserpine ------N = 15 OEIEHIE moles/liter) (m NOREPINEPHRINE 1 ------

HRCC AORTA THORACIC xIO 1 ------9 - 1 - - - - -

D0=.7+2. xIO-9 4 .0 2 + =4.07 ED50 ------1 - - - - - 1- FET F EEPN PERAMN ON PRETREATMENT RESERPINE OF EFFECT Control N = 39 ------ACLR RESPONSES NE TOVASCULAR

1 ------1 -• CONTROL -• ■oRESERPINE 100 0 4 0 2 60 80-1 H IO OEIEHIE moles/liter) (m NOREPINEPHRINE EDS0=5.65+I.67xl0"8 *12 Reserpine IO N=I5 EETRC ARTERY MESENTERIC '11 IO -10 IO *9 IO D0=5. . 0-8 l0 x 4 i.6 3 .5 5 ED50= *8 IO Control "7 N=35 IO *6

IO *5 o 71 alpha receptor or a receptor similar to the alpha receptor. If this were correct then increasing concentrations of phentolamine should pro duce a parallel shift to the right of the concentration response curve for serotonin. Furthermore, the inhibitory effect of phentolamine on serotonin-mediated contraction should occur with concentrations of phentolamine that were similar to those concentrations required for inhibition of the contractile responses to norepinephrine. These postulates' were tested employing the criteria of Furchgott (see Methods and Materials section). The results are summarized in Figures 9 through

11 and Table 3.

The contractile responses of rat thoracic aortae to serotonin were shifted to the right in a parallel fashion (P>0.05) thirty minutes after the addition of phentolamine (1 X 10“% ) to the muscle strips. However, a ten fold increase in the concentration of this alpha adrenoceptor blocking agent did not effect a further inhibition of serotonin-mediated

contractions of rat thoracic aortae. A slight further inhibition of serotonin

responses was observed with 10~6m phentolamine whereas the responses to

serotonin were almost abolished thirty minutes after the addition of

10“% phentolamine to the muscle strips. Similar findings were observed

in the mesenteric artery. However, lCT^M phentolamine produced a more

pronounced inhibition of the contractile responses to serotonin than had •

been effected in the thoracic aorta. In contrast to these findings,

phentolamine produced the anticipated parallel shift to the right of the

concentration effect curves for norepinephrine. Although the maximal

response to norepinephrine was depressed, which would suggest a non- TABLE 3

pA2 VALUES OF METHYSER.GIDE AND PHENTOLAMINE AGAINST NOREPINEPHRINE AND SEROTONIN ON RAT THORACIC AORTAE AND MESENTERIC ARTERIES TABLE 3. pA2 VALUES OF METHYSERGIDE AND PHENTOLAMINE AGAINST NOREPINEPHRINE AND SEROTONIN ON RAT THORACIC AORTAE AND MESENTERIC ARTERIES

THORACIC ARTERY MESENTERIC ARTERY

TREATMENT NE 5-HT NE 5-HT

(pA2-log moles/Liter) ± S.E.M.

Phentolamine Control 9.35 ± 0.03 9.1 ± 0.2 9.85 ± 0.35 8.55 ± 0.70**

Phentolamine s ^ i o -Sm 8.83 ± 0.07* 7.85 ± 0.2* 8.49 ± 0.12* 8.7 ± 0.15

Phentolamine Reserpine 8 . 8 ± 0 .1* 9.75 ± 0.35** 8.9 ± 0 .2* 10.7 ± 0.4**

Methysergide None 8.65 ± 0.30 9.5 ± 0.25** 7.5 ± 0.3* 12.13 ± 0.22**

Methysergide Reserpine 8.1 ± 0.2* 11.0 ± 1.0** 7.85 ± 0.4* 11.6 ± 0.85**

Animals were pretreated with reserpine (2.5 mg/kg l.p.) for six days prior to experimentation. * Responses differ significantly (P<0.05) from corresponding control. ** Responses differ significantly (P<0.05) from NE.

pA2-concentration of antagonist necessary to increase the ED^q concentration of agonist by a factor of two. Each value represents the mean ± S.E.M. of four experiments each obtained from individual vascular strips. 74

FIGURE 9

EFFECT OF PHENTOLAMINE ON VASCULAR SMOOTH MUSCLE RESPONSES TO NOREPINEPHRINE AND SEROTONIN

Effect of phentolamine on the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin and norepinephrine. The ordinate represents the contractile responses of the vascular strips to the agonists (expressed as a percentage of the maximum control responses to the agonist). The abscissa represents the molar concentration of agonist. Standard errors did not exceed 7 percent. Each mean value represents the responses from four vascular strips, each vascular strip representing one rat. The antagonist was in contact with the muscle strips for thirty minutes prior to evaluation of the responses to the agonist. CONTRACTILE RESPONSE (% MAXIMUM) lOO-i lOO-i 40 60- 80- 40- 60- 0 2 80- 20 0 0 - - - O1 I' I' I 7 IO*6 '7 IO IO'8 IO'9 IO'10 O 0 O9 O8 07 IO'6 I0‘7 IO*8 IO'9 IO’ 10 T MOH UCE EPNE T NRPNPRN AD SEROTONIN AND NOREPINEPHRINE TO RESPONSES MUSCLE SMOOTH • Control• Phentolamine N =4 5-HT(moles /liter) 5-HT(moles -T (moles/liter) 5-HT T TT FET F HNOAIE N VASCULAR ON PHENTOLAMINE OF EFFECT T

IO'5 T 10 MESENTERY " AORTA o I00-, 100-1 40- 40- 80- 20 80- 60- 60- 20 - - OEIEHIE (moles/liter) NOREPINEPHRINE 10*1° OEIEHIE (moles/liter) NOREPINEPHRINE iq 9 O8 O7 O6 IO'5 IO'6 IO'7 IO"8 -9 08 O7 O6 IO'5 IO*6 IO'7 I0*8

B 75 76

FIGURE 10

EFFECT OF COCAINE (5 X 10-6 M) ON PHENTOLAMINE MEDIATED INHIBITION OF THE CONTRACTILE RESPONSES TO SEROTONIN

Effect of neuronal reuptake blockade with cocaine (5 X 10”% ) on phentolamine mediated inhibition of the contractile responses to serotonin. The ordinate represents the contractile force of the muscle generated upon addition of serotonin to the muscle bath (expressed as a percentage of the maximal response of the control response). The abscissa represents the molar concentration of serotonin. Vertical lines are the standard errors of the means. Each mean value represents the responses from four vascular strips representing four animals. Cocaine was in contact with the muscle strips throughout the entire experiment.

Each concentration of phentolamine was allowed to equilibrate with the muscle strips for thirty minutes prior to evaluation of the degree of serotonin inhibition. CONTRACTILE FORCE (%OF MAXIMUM ) t SEM lOOn 60- 80- 0 2 40- - o—o FET F OAN (xC6 O PET A N MEDIATED INE LAM PHENTO ON ) (5xlCT6M COCAINE OF EFFECT 0' I' I" I" IO-5 IO"6 IO"7 IO'8 '9 I0 N = 4 NIIIN F H CNRCIE EPNE T SEROTONIN TO RESPONSES CONTRACTILE THE OF INHIBITION Ccie(xO6M) Cocaine(5xIO"6 • P<0.05 * Petlmn (0'M) '8M (I0 Phentolamine + I _7M) (I0 OT MSNEI ARTERY MESENTERIC AORTA

EOOI ( es/ier) /lite s le o (m SEROTONIN lOO-i 40- 60- 20 80- - 09 I" I' I" IO’ 5 IO"6 IO'7 IO"8 • 10"9 N = 4

**4 78

FIGURE 11

EFFECT OF PHENTOLAMINE ON RESPONSES OF VASCULAR SMOOTH MUSCLE FROM RESERPINE (2.5 mg/Kg i.p.) PRETREATED RATS TO NOREPINEPHRINE AND SEROTONIN

Effect of reserpine pretreatment (2.5 mg/kg/day for six consecutive days) on phentolamine mediated inhibition of the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin arid norepi nephrine. The ordinate represents the contractile responses of the vascular smooth muscle to the agonists (expressed as a percent of maximum response). The abscissa represents the molar concentration of agonist.

Standard errors did not exceed ± 5 percent. Reserpine was administered i.p. Phentolamine was allowed to equilibrate with the vascular strips for thirty minutes prior to evaluation of the contractile responses to the agonists. 79

EFFECT OF PHENTOLAMINE ON RESPONSES OF VASCULAR SMOOTH MUSCLE FROM RESERPINE (2.5m g/Kg i.p.) PRETREATED RATS TO NOREPINEPHRINE AND SEROTONIN AORTA

120-1 A •CONTROL lOO-i • PHENTOLAMINE I0‘ 8 100- • IO'7 80- • IO'6 80- o IO'5 60- 60-

40- 40-

20 - 20

0 -

to-12 KT" IO'10 IO’9 IO'8 IO'7 ICT6 IO-5 icr 12 k t " io '10 io'9 icr 8 io-7 ior® icr 5 5-HT (moles/liter) NOREPINEPHRINE (moles/liter)

MESENTERY

C D 100- IOO-i

60- 80

60 60

40- 40- N *4 N * 4

20 - 20 -

T T

5-HT (moles/liter) NOREPINEPHRINE (moles/liter) 80 competitive or irreversible blockade of high doses of norepinephrine with phentolamine, I cannot at present offer an explanation for this

finding. The maximal response to norepinephrine is only depressed

approximately 1 0 percent during the time course of this study (see

Figure 4A and 5A). Thus as summarized in Figure 8 , phentolamine produces

a complex blockade of the contractile responses to serotonin, and in part to norepinephrine. However, concentrations of phentolamine

(1 X 10“% ) just necessary to inhibit the responses of rat mesenteric

arteries and thoracic aorta to norepinephrine inhibit the contractile

responses to serotonin.

The studies of Jarrot, et at, (1975) and Berkowitz, et aly (1972)

suggest that serotonin may be stored within the thoracic aorta and

mesenteric arteries of the rat. It was possible that phentolamine may

not only interact with the smooth muscle receptor for serotonin but with

other binding sites as well. This could result in an increased seroto

nin concentration at the receptor site overcoming the blocking acvitity

of phentolamine resulting in the concentration effect curves demonstrated

in Figure 9. Therefore I repeated the previous studies in vascular strips

incubated with cocaine (5 X 10"%) to inhibit the active reuptake mecha

nisms. The results are summarzied in Figure 10. Phentolamine produced

concentration related parallel shifts to the right of the concentration

effect curves of both thoracic aortae and mesenteric arteries to serotonin.

The concentration of phentolamine necessary to inhibit serotonin mediated

contraction was increased ten fold in rat thoracic aorta. Similar

results were observed for norepinephrine (Table 3). This, blockade of 81 some yet undefined reuptake mechanism for serotonin appears to convert phentolamine-mediated inhibition of the contractile responses to seroto nin from a mixed type to a competitive type of inhibition (Figure 9).

The above findings suggested that phentolamine may act on an alpha like receptor to inhibit serotonin mediated contraction of rat vascular smooth muscle. Were this concept correct then endogenous norepinephrine and serotonin may compete with phentolamine for these receptors thus diminishing the blocking potency of phentolamine. Reserpine was there fore employed in an attempt to deplete the vascular smooth muscle cells of norepinephrine, as well as serotonin. The results are summarzied in

Figure 11. In thoracic aortae obtained from reserpine pretreated rats, phentolamine inhibited the responses to serotonin, similar to that observed in muscles obtained from control animals (compare Figure 9 and

Figure 11). However, the magniture of initial blockade and final block ade (1 0 - % phentolamine) was greater than that observed in control strips (P>0.05). Similarly, phentolamine-mediated inhibition of serotonin-induced contraction of rat mesenteric arteries was greater than that observed in mesenteric arteries obtained from untreated control rats (Figure 9 and Figure 11). Phentolamine-mediated inhibition of norepinephrine-induced contractions of rat vascular smooth muscle was not enhanced from control values (Figure 11 and Table 3) but actually was less effective (P<0.05) an antagonist of norepinephrine-induced contractions of rat thoracic aortae and mesenteric arteries. These findings suggest that in the presumed absence of endogenous norepinephrine 82

and serotonin, phentolamine is a more effective antagonist of serotonin

than of norepinephrine. This further suggests that serotonin is

eliciting contraction through stimulation of an alpha-receptor or a

receptor so similar to that of the alpha receptor that phentolamine

cannot distinguish between the two classes of receptors. This postulate was examined further.

Tolazoline, a purportedly less selective, less potent adrenoceptor

antagonist than phentolamine (Goodman and Gilman, 1975), was utilized as the

second alpha adrenoceptor antagonist. The data are summarized in Figures 12 and 13. The maximal contractile responses of the rat thoracic aortae and mesenteric arteries to serotonin were slightly but significantly depressed

by tolazoline. The remainder of the concentration response curve was

unaffected by this alpha adrenoceptor antagonist. In contrast, tolazo

line produced a competitive, parallel shift to the right of the

concentration response curves for norepinephrine (Figure 12). The depres

sion of the maximal responses to norepinephrine was not related to the

concentration of tolazoline employed in the studies and was slightly more

than the depression observed in the vascular strips with repeated concen

tration effect curves to this amine (Figure 4). In the presence of amine

depletion and neuronal reuptake blockade with reserpine and cocaine re

spectively (Figure 13), tolazoline appeared to act as a partial agonist

of the serotonergic receptor since it enhanced the responses to low con

centrations of serotonin whereas, similar to that observed in the previ

ous experiment (Figure 12), the maximal contractile response to serotonin

was slightly depressed. However, the aortic responses are difficult to 83 interpret since tolazoline contracted the aortic strips (Figure 14).

This should have resulted in a depression of the responses to serotonin.

It is apparent from Figure 13 that tolazoline only exerted a minimal in hibitory effect on the contractile responses to this indole-alkylamine.

According to Van Rossum (1963), a pure noncompetitive antagonist will produce a depression of the maximum response to the agonist provided spare or reserve receptors exist within the muscle preparation for the agonist.

Tolazoline did not affect the contractile responses of the rat vascular smooth muscle to serotonin, when obtained from normal animals. However, in the absence of endogenous amines and neuronal reuptake blockade, tolazoline appears to act as a partial agonist (Figure 13) and as a non competitive antagonist of serotonin. Under these conditions tolazoline is still a competitive antagonist of norepinephrine (Furchgott, 1972).

These data add further support to the concept that serotonin-mediated contraction of rat vascular smooth muscle may be initiated through alpha receptors which differ from that of the classical alpha adrenergic receptor.

Effect of Serotonergic Receptor Blockade on the Contractile

Responses of Rat Thoracic Aortae and Mesenteric Arteries to

Serotonin and Norepinephrine

Methysergide was a more potent inhibitor of the contractile responses to serotonin than to norepinephrine (Figure 15). However, methysergide produced a concentration related, competitive blockade of the contractile responses to norepinephrine as well. Similar 84

FIGURE 12

EFFECT OF TOLAZOLINE ON VASCULAR SMOOTH MUSCLE RESPONSES TO NOREPINEPHRINE AND SEROTONIN

Effect of tolazoline on the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin and norepinephrine. The ordinate represents the contractile responses of the vascular strips to the agonists (expressed as a percentage of the maximum control responses to the agonist). The abscissa represents the molar concentration of agonist. Standard errors did not exceed 7 percent. Each mean value represents the responses from four vascular strips, each vascular strip representing one rat. The antagonist was in contact with the muscle strips for thirty minutes prior to evaluation of the responses to the agonist. CONTRACTILE RESPONSE (% M A X IM U M ) lOO-i lOO-i - 0 4 - 0 6 80- 0 2 - 0 4 20 80- 0 6 A C 0 0 - - - - OAOIE O 8 IO’ TOLAZOLINE * CONTROL ' • a T T 1 io'1 r10 FCT F OL OLNE N ACLR MOH MUSCLE SMOOTH VASCULAR ON E LIN ZO LA TO OF T FFEC E 10 T * -T( lsltr NRPNPRN ( oles/liter) (m NOREPINEPHRINE oles/liter) (m 5-HT -T moe/ie) OEIEHIE moles/liter) (m NOREPINEPHRINE oles/liter) (m 5-HT 7 EPNE T NOREI P NE N SEROTONIN AND E IN R EPH EPIN R O N TO RESPONSES

T MESENTERY AORTA lOO-i lOO-i - 0 6 20 - 0 8 - 0 4 - 0 4 - 0 6 20 B 80- - - 0' I' I' I' I' I '5 IO IO'6 IO'7 IO'8 IO'9 10*'° O1 I' I' I' I* I '9 IO IO*6 IO'7 IO'8 IO'9 IO*10

85 86

FIGURE 13

EFFECT OF NEURONAL REUPTAKE BLOCKADE AND AMINE DEPLETION ON TOLAZOLINE-MEDIATED INHIBITION OF SEROTONIN

Effect of reserpine pretreatment (2.5 mg/kg/day for six days) and neuronal reuptake blockade with cocaine (5 X 10"^M) on tolazoline- mediated inhibition of the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin. The ordinate represents the contractile responses of the vascular smooth muscle to serotonin

(expressed as a percent of maximum response). The abscissa represents the molar concentration of serotonin. Standard errors did not exceed the mean value ± 6 percent. Each value represents the mean response obtained from four vascular strips. Each vascular strip was obtained from an individual animal. Reserpine was administered i.p. Cocaine was present in the muscle bath throughout the experimental procedures.

Tolazoline was equilibrated with the vascular strips for thirty minutes prior to evaluation of the responses to serotonin. An asterisk denotes the responses to serotonin in the presence and absence of tolazoline significantly (P<0.05) differ. CONTRACTILE FORCE (% OF CONTROL) DEPLETION AMINE AND BLOCKADE REUPTAKE NEURONAL OF EFFECT IOO- 40- 60- 20 80- - i N OAOIEMDAE IHBTO O SEROTONIN OF INHIBITION TOLAZOLINE-MEDIATED ON O9 O8 O7 O6 IO*5 IO'6 IO"7 IO'8 IO'9 o—o (IO"8M) Tolazoline • Control • HRCC OT MSNEI ARTERY MESENTERIC AORTA THORACIC I '7M) (I0

N= 4 EOOI (oe/lt r) lite (moles/ SEROTONIN OO-i - O lO 40- 0 2 60- 80- - O9 O' I' I' I '5 IO IO'6 IO'7 '8 IO IO'9 N= 4

FIGURE 14

EFFECT OF TOLAZOLINE ON VASCULAR SMOOTH MUSCLE TONE

Effect of tolazoline on the basal force of rat thoracic aortae and mesenteric arteries. The ordinate represents the contractile response of the vascular strips to tolazoline (milligrams). The abscissa repre sents the molar concentration of tolazoline. Vertical lines are the standard errors of the means. Each value- represents the responses of four vascular strips obtained from individual rats. Tolazoline was in contact with each vascular strip for thirty minutes prior to measurement of force. CONTRACTILE FORCE (mg)t SEM FETO TLZLN O VSUA SOT MSL TONE MUSCLE SMOOTH VASCULAR ON TOLAZOLINE OF EFFECT 300- 400- 500-i 200 0 0 1 - - . as rtetd o sixdays for pretreated Rats I. Ccie(xO6M inbath M) Cocaine(5xlO"6 . with Reserpine (2.5mg/Kg/day i.p.) (2.5mg/Kg/day Reserpine with OAOIE (moles/liter) TOLAZOLINE AORTA (4) EETRC REY (4) ARTERY MESENTERIC ■o 90 findings were observed in vascular strips depleted of their amine stores by chronic treatment with reserpine (Figure 16). Following amine deple tion with reserpine, methysergide was a more potent inhibitor of the contractile responses to serotonin than in vascular strips obtained from control animals. Similarly, amine depletion decreased the noradrenergic blocking activity of methysergide. Nevertheless, cross reactivity between these two receptors is also apparent with classical serotonergic receptor antagonists.

Effect of Blockade of Tolazoline Sensitive Receptors on Phentolamine and Methysergide Mediated Inhibition of the Contractile Responses of Rat Vascular Smooth Muscle

to Serotonin and Norepinephrine

The previous experiments demonstrated that alpha and "serotonergic receptor" antagonists appear to have cross sensitivities to norepine phrine and serotonin. This is not shared by the alpha adrenoceptor blocking agent, tolazoline. If serotonin were acting on an alpha recep

tor different from that of the classical alpha receptor, then blockade of

the putative classical receptors with tolazoline should unmask the

second type of receptor and allow for a quantitative analysis of its

kinetic properties. This postulate was examined. The results are

summarized in Figures 17, 18, and 19. Tolazoline (1 X 10“% ) and cocaine

(5 X 10-%) were employed to block the purported classical alpha receptor

and the neuronal reuptake of norepinephrine and serotonin which would

interfere with the results of the study. Vascular strips were 91

FIGURE 15

EFFECT OF METHYSERGIDE ON VASCULAR SMOOTH MUSCLE RESPONSES TO NOREPINEPHRINE AND SEROTONIN

Effect of serotonergic receptor blockade with methysergide on the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin and norepinephrine. Methysergide was equilibrated with the muscle strips for thirty minutes prior to re-evaluation of the responses to serotonin and norepinephrine. The ordinate represents the contractile responses of the vascular strips to the agonists (expressed as a per centage of the maximum control responses to the agonist). The abscissa represents the molar concentration of agonist. Standard errors did not exceed 7 percent. Each mean value represents the responses from four vascular strips, each vascular strip representing one rat. CONTRACTILE RESPONSE (% MAXIMUM) IOO-J IOO- 40- 60 40- 20 80 20 80- 60- A C 0 0- - - - i | -7 |0 a IO"8 Methysergide * • Control • IQ '6 IO □ ■ O1 I’9 O8 I" IO'6 IO"7 IO-8 9 IO’ IO'10 01 1- 1- I- IO-6 IO-7 10-8 10-9 10-10 4 =N - moles/liter) (m T 5-H *8 -T moles/liter) (m 5-HT MESENTERY AORTA FET F EHSRIE N ACLR SMOOTH VASCULAR ON METHYSERGIDE OF EFFECT UCE EPNE T NRPNPRN AND NOREPINEPHRINE TO RESPONSES MUSCLE T

io- IO-® EOONIN SEROTO 0 0 1 120-1 I00-, 40- 80- 40- 60- 60- 20 80- B 20 - - - io N»4 N=4 *10 OEIEHIE moles/liter) (m NOREPINEPHRINE OEIEHIE moles/liter) (m NOREPINEPHRINE 10 lo iq -9 9 O8 I" I" IO"5 IO"6 IO"7 IO-8 -9 MESENTERY io AORTA -8 io

-7 io

'6 cr ic 92 5 93

FIGURE 16

EFFECT OF METHYSERGIDE ON RESPONSES OF VASCULAR SMOOTH MUSCLE FROM RESERPINE (2.5 mg/Kg i.p.) PRETREATED RATS TO NOREPINEPHRINE AND SEROTONIN

Effect of reserpine pretreatment (2.5 mg/kg/day for six consecutive days) on methysergide-mediated inhibition of the contractile responses of rat thoracic aortae and mesenteric arteries to serotonin and norepi nephrine. The ordinate represents the contractile responses of the vascular smooth muscle to the agonists (expressed as a percent of maxi mum response). Methysergide was allowed to equilibrate with the vascular strips for thirty minutes prior to re-evaluation of the responses to agonists. Reserpine was administered i.p. Standard errors did not exceed the mean value ± 6 percent. CONTRACTILE RESPONSE (% MAXIMUM) lOOi IOO- - 0 4 - 0 6 0- 4 60- 80- 80- 20 0 2 A OH FET F EHSRIE N EPNE O VSUA SOT MUSCLE SMOOTH VASCULAR OF RESPONSES ON METHYSERGIDE OF EFFECT - i EHSRIE 10"8 METHYSERGIDE • a 0‘6 I0 • ••CONTROL d Ol | 9 O8 O7 O6 IO'5 IO'6 IO'7 IO'8 '9 |0 IO'l° O1 I‘9 O8 O I' IO'5 IO'6 7 IO’ IO'8 9 I0‘ IO'10 O'5 IO IO"7

HT ( oles/liter) (m T -H 5 RM EEPN (2. Kg ip) RTETD RATS PRETREATED i.p.) g /K g m .5 2 ( RESERPINE FROM *T moles/liter) (m 5*HT O NEPHRI N SEROTONIN AND E IN R H P E IN P E R O N TO

MESENTERY AORTA

I00-, IOO-i 0 6 - 0 4 20 60- 80- BO- 40- 20 B 0 - - - N* 4 N* NOREPINEPHRINE (moles/liter) NOREPINEPHRINE OEIEHIE( oles/liter) (m NOREPINEPHRINE

94 95 equilibrated with these two antagonists for three hours prior to and throughout the experiment. As summarized in Figure 17A (left panel), phentolamine (1 X lCT^M) inhibited the contractile responses of the rat thoracic aorta to serotonin. Lower concentrations appear to enhance the contractile responses to this indole-alkyl amine. In contrast, phentol amine did not affect the residual responses to norepinephrine, while demonstrating an apparent enhancement of the responses to the catechol amine (Figure 17B, right panel). Similarly, in vascular strips treated with tolazoline, methysergide produced a concentration dependent inhibi tion of the contractile responses to serotonin (Figure 18A, left panel) but did not inhibit the contractile responses of rat thoracic aortae to norepinephrine (Figure 18B, right panel). The apparent enhancement of the responses to serotonin and norepinephrine with these two antagonists

is not apparently mediated by the blocking agents. As demonstrated in

Figure 19, the contractile responses of the rat thoracic aortae to

serotonin and norepinephrine are tremendously enhanced during the course of the experiment in the absence of either methysergide or phentolamine.

This is in contrast to the very stable responses observed in normal vascular strips which do not undergo prolonged incubation with tolazo

line. This effect is not due to the cocaine since the responses to

these agonists are stable with prolonged exposure to this inhibitor of

neuronal reuptake blockade. The significance of this phenomenon will

be reviewed in the discussion. However, the data clearly demonstrate

that phentolamine will inhibit more selectively the contractile

responses to serotonin than those to norepinephrine when tolazoline

is employed to inhibit alpha 96

FIGURE 17

EFFECT OF PHENTOLAMINE ON THE VASCULAR SMOOTH MUSCLE RESPONSES TO NOREPINEPHRINE AND SEROTONIN IN RAT THORACIC AORTA IN THE PRESENCE OF TOLAZOLINE PLUS COCAINE

Effect of alpha receptor blockade with tolazoline and neuronal reuptake blockade with cocaine (5 X 10“% ) on phentolamine mediated inhibition of the contractile responses of rat thoracic aortae to serotonin and norepinephrine. The ordinate represents the contractile responses of the vascular strips to the agonists (expressed as a percentage of maximal control responses). The abscissa represents the molar concentration of agonist. Vertical lines are the standard errors of the means. Each value represents the responses obtained from four vascular strips representing four rats. Asterisks denote that the responses in the presence and absence of phentolamine significantly dif fer (P<0.05). Tolazoline (1 X 10"%) and cocaine (5 X 10“% ) were present in the muscle bath throughout the three hour equilibration period as well as throughout the entire experiment. Phentolamine was allowed to equilibrate with the muscle strips for thirty minutes prior to re- evaluation of the responses to the agonist. Note well the presence of responses to norepinephrine and serotonin during the control concentra

tion response curve. CONTRACTILE FORCE (V.MAXIMUM)t SEM 220 - 0 4 2 280-1 200 260- 140- 0 0 1 180- 160- 120 - 0 4 20 80- 60- 0 ------O1 I* I' I* I* I* XI0*9 IO*83X IO*6 IO*7 IO'9 IO*9 IO*10 ■ A A a CONTROL • □ IFR SGIIATY RM OTO (P<0.05) CONTROL FROM SIGNIFICANTLY DIFFERS * OTO 2. 35XO*7 XIO 5 .3 0 + 3 .3 2 CONTROL 0* PHENTOLAMINE *s I0 P<0.05 10 10 N=4 10 10 PHENTOLAMINE LS OAN ( 0*6M PIR O N HOGOT H ORE F H EXPERIMENT THE OF COURSE THE THROUGHOUT AND TO PRIOR M) 6 * I0 X (5 COCAINE PLUS ISE WR ALWD O QIIRT FR HE HUS N S CNANN TLZLN (I0' M) '4 0 I ( TOLAZOLINE CONTAINING PSS IN HOURS THREE FOR EQUILIBRATE TO ALLOWED WERE TISSUES .1X1“® X10“ 0.91 i 5 7 . 4 * * * " " 6 6 s 7 9

SEROTONINt MOLES/LITER) SEROTONINt d e 50

O NEPHRI IN N O T O R E S D N A E IN R H P E IN P E R O N TO S E S N O P S E R E L C S U M F NE O THE VASCULAR SMOOTH O O M S R A L U C S A V E H T ON E IN M A L O T N E H P OF T C E F F E

IN RAT THO RACIC AO RTA IN T H E PRESENCE OF PRESENCE E H T IN RTA AO RACIC THO RAT IN NE PUS AN ** * CAINE O C S PLU E IN L O Z A L O T

Ti bJ o UJ 2 180- S

- 0 4 2 280-1 ‘ 0 0 2 220 260- 0 0 1 140- 120 160- - 0 4 0- 8 20 60- - - - - O9 PHENTOLAMINE IO*9 IO-1 X 0.9 t 4.i CONTROL 09 O9 O7 O6 O9 IO'43XI0*4 IO*9 IO*6 IO*7 IO*9 I0*9 P<0.05 * 5+2. XIO*6 .4 2 + .5 5 N * 4 * N OEIEHIE MOLES/LITER) ( NOREPINEPHRINE 90 d e

\D 98

FIGURE 18

EFFECT OF METHYSERGIDE ON THE VASCULAR SMOOTH MUSCLE RESPONSES OF RAT THORACIC AORTA TO SEROTONIN AND NOREPINEPHRINE IN THE PRESENCE OF TOLAZOLINE (1 X 10"%) AND COCAINE (5 X 10“% )

Effect of alpha receptor blockade with tolazoline and neuronal reuptake blockade with cocaine (5 X 10“% ) on methysergide-mediated inhibition of the contractile responses of rat thoracic aortae to serotonin and norepinephrine. The ordinate represents the contractile responses of the vascular strips to the agonists (expressed as a percentage of maximal control responses). The abscissa represents the molar concentration of agonist. Vertical lines are the standard errors of the means. Each value represents the responses obtained from four vascular strips representing four rats. Asterisks denote that the responses in the presence and absence of methysergide significantly dif fer (P<0.05). Tolazoline (1 X 10 % ) and cocaine (5 X 10"%) were present in the muscle bath throughout the three hour equilibration period as well as throughout the entire experiment. Methysergide was allowed to equilibrate with the muscle strips for thirty minutes prior to re- evaluation of the responses of the thoracic aortae to the agonists. CONTRACTILE RESPONSE (% MAXIMUM + SEM ) 100-1 - 0 8 - 0 4 20 - 0 6 160- - 0 8 0 - - ACLR TIS EE LOE T EULBAE O 3 OR I PS LS 0‘4 TOLAZOLINE M 4 ‘ I0 PLUS PSS IN HOURS 3 FOR EQUILIBRATE TO ALLOWED WERE STRIPS VASCULAR * LS 06M OAN PIR O N TRUHU TE ORE F H EXPERIMENT THE OF COURSE THE THROUGHOUT AND TO PRIOR COCAINE M I0'6 X 5 PLUS EPNE O RT HRCC OT T SRTNN N NOREPINEPHRINE AND SEROTONIN TO AORTA THORACIC RAT OF RESPONSES in-7 CONTROL IO’ 10 IO'® N=2 METHYSERGIDE N H PEEC O TLZLN (I0' AD OAN (XI 6M)** * ) M T6 IC (5X COCAINE AND ) M '4 0 I ( TOLAZOLINE OF PRESENCE THE IN * * 9 EOOI ( OLES/LITER) (M SEROTONIN FET F EHSRIE N H VSUA SOT MUSCLE SMOOTH VASCULAR THE ON METHYSERGIDE OF EFFECT OTO 07 .5 I’7 IO’ X 0.25 + 0.74 CONTROL EDso r® 7 r O 93XI0*9 X IO’93 +i - 0 4 2 2 - 0 4 o o V) 2 t o L l I

200 220 - 0 6 2 180- 0 0 1 120 140- 0 2 B 0 ------O8 I’ I' IO*9 IO'6 IO’7 . IO"8 * r OEIEHIE MOLES/LITER) O (M NOREPINEPHRINE =2 = N OTO 2. 1.1XIO"6 i .6 2 CONTROL .. . . a * . . i. > —_ ...... — _ > . i . . . d e 50

VO vo 1 0 0

FIGURE 19

TEMPORAL RESPONSES OF TOLAZOLINE IN RAT THORACIC AORTA TO NOREPINEPHRINE AND SEROTONIN IN THE PRESENCE OF COCAINE

Temporal stability of rat thoracic aortae to norepinephrine and serotonin during continual incubation with tolazoline (1 X 10“^M) and cocaine (5 X 10” M). The ordinate represents the contractile responses to serotonin and norepinephrine after the three hour equilibration peri od and four hours later (final response). The data are expressed as a percent of the maximal contractile response of the muscle to the agonist

(initial response). The abscissa represents the molar concentration of agonist. Vertical lines are the standard errors of the means. Each mean value represents the responses from three vascular strips each obtained from an individual rat. An asterisk denotes that the initial and final responses significantly differ (P<0.05) from each other.

Cocaine and tolazoline were present in the physiologic saline solution during the three hour equilibration period and throughout the entire

experiment. CONTRACTILE FORCE ( % MAXIMUM)* SEM 200-, 0 0 1 140- 180- 160- 120 - 0 4 - 0 6 0- 8 20 • SSUES WR ALWD O QIIRT FR HUS N S CNANN TLZLN (0*4M) 4 * (I0 TOLAZOLINE CONTAINING PSS IN HOURS 3 FOR EQUILIBRATE TO ALLOWED WERE S E U S IS ••T - - - FFERS SGNII L TLY N A IFIC N SIG S R E F IF D • LS OAN ( 0* PIR O N TRUHU TE ORE F H EXPERIMENT THE OF COURSE THE THROUGHOUT AND TO PRIOR ) M *® I0 X (5 COCAINE PLUS nto 3.3+I33XI *® IO X 3 I.3 + .I3 3 Initiol —0 0— 0® O7 0® O9 043XI0"4 I0*4 IO*9 10*® IO*7 10*® P<0.05 * ia 0. 02 XIO*® 0.26 i 8 .7 0 Final O NEPHRI E ONN I F NE * * E IN A C O C OF E C N E S E R P E H T IN NIN TO O SER D N A E IN R H P E IN P E R O N TO NOREPINEPHRINE F NE I RAT C AORTA IC C A R O H T T A R IN E IN L O Z A L O T OF S E S N O P S E R L A R O P M E T

IA RESPONSE FINAL NTA RESPONSE INITIAL EDso MOLAR CONCENTRATION OF AGONIST OF CONCENTRATION MOLAR ( P<0.05) P<0.05) (

R CONTROL M FRO o o z h- t- o a: o uj 5 s c

- 0 4 - 0 6 80 20 B 0

- - - - - P <0.05 * nto 1.8 .8XO*7 XIO 0.58 .38+ 1 Initiol Final 0.183 + 0.07 XIO*7 0.07 + 0.183 Final O' 10*® '9 IO

SEROTONIN ED 10 S0 -r "1 0 6 I0‘

“n IO*9

101 1 0 2 adrenoceptors. Furthermore, methysergide becomes a specific antagonist of serotonin upon blockade of tolazoline sensitive receptors. These data lend further support to the concept that the serotonergic receptor may be similar to, but distinct from, the classical alpha adrenergic receptor.

Semiquantitative Analyses of the Adrenoceptor

Antagonist-Serotonin Interaction

Table 3 summarizes the concentration of antagonist necessary to increase the ED5 0 of the respective agonist by a factor of two. The derived value, pA£, is characteristic of a specific antagonist-receptor interaction. If the pA£ values are similar and the slope of the

Arunlakshana and Schild plot of Log (dose ratio-1) vs Log Molar

Concentration of Antagonist are equal to 1 then for two agonists, interacting with the same antagonist, the data are interpreted to indi cate the existence of a single receptor. If the pA£ values differ and the slopes of the plot are similar, the data suggest that the receptors may differ. The slopes and pA£ values for the antagonists against norepinephrine were constructed as described in the methods section.

This posed no difficulty since phentolamine produced a parallel shift to the right on the concentration effect curve for norepinephrine. However, as evidenced by Figures 9, 10, and 11, this was more complex for serotonin. Thus the best fitting straight line was obtained for the con centrations of the antagonists that produced inhibition of the responses to serotonin. The data obtained are not true pA£ values but merely estimates of the real values. The slopes of the interactions shown did not deviate 103

significantly from each other nor from the theoretical value of 1

(Furchgott, 1972). The pA2 values differ significantly from each other for serotonin and norepinephrine. These findings clearly support the concept that the serotonergic receptor is a phentolamine sensitive receptor which differs from that of the typical alpha adrenergic receptor.

Effect of Calcium Ion on Phentolamine Mediated

Inhibition of Norepinephrine

Acceptance of the above findings and their suggestive conclusions implies the acceptance of the validity of the conditions of the experi ments and the results obtained with the adrenergic receptor stimulant norepinephrine when challenged with phentolamine. The pA£ (Table 3, page 73) value of phentolaraine-induced antagonism of norepinephrine is more than one log unit greater than that previously reported (Furchgott,

1967; Furchgott, 1972). If this pA2 is incorrect, then the validity of the entire study is in doubt. Almost all studies evaluating phentolamine-mediated antagonism of norepinephrine have employed an ionized calcium concentration of 2.0 to 2.5 mM in PSS (Innes and Kohli,

1970; Furchgott, 1954; Vanhoutte, et a l , 1969). The physiologic ionized calcium concentration of plasma appears to be approximately

1.5-1.8 mM when the protein bound fraction of calcium is eliminated from measurement (Greenberg, et at, 1973). All experiments were per formed with an ionized calcium concentration of 1.6 mM. Therefore, the experiments were repeated using the same PSS employed in the 104 initial studies except that the calcium concentration was increased to

2.5mM. The results are summarized in Figure 20. Phentolamine produced a concentration related inhibition of the contractile responses to norepinephrine. While the sensitivity to norepinephrine itself was not affected by the increased calcium ion concentration, the blocking activity of phentolamine was shifted to the right by a factor of 1 log unit. The slope of the Log (dose ratio-1) versus the log concentration of phentolamine did not deviate significantly from parallelism in vascular strips exposed to 1.6 and 2.5 mM calcium ion. The pA£ value obtained with PSS containing 2.5 mM calcium ion (8.21) is in complete agreement with the studies of most investigators (for references see

Furchgott, 1972). Thus, the higher pA£ values (i.e., enhanced adreno ceptor blocking activity of phentolamine) observed in our studies appears to be related to the more physiologic calcium concentration employed in these studies. A detailed analysis of the interaction between calcium ion and the 5-HT and norepinephrine blocking activity of adrenoceptor blocking agents is beyond the scope of this study and will be reported in detail (Curro, et al, 1976).

Binding of 2-C-^-Serotonin to Rat Thoracic Aorta

The above studies suggested that phentolamine may inhibit the binding of serotonin to its receptor site on the vascular smooth muscle cell membrane. Were this postulate correct then the binding of radiolabeled serotonin to isolated strips of rat thoracic aortae should be diminished following pre-incubation of vascular strips with phentolamine. This FIGURE 20

EFFECT OF CALCIUM ION ON PHENTOLAMINE ANTAGONISM OF NOREPINEPHRINE ON RAT AORTA

Effect of calcium ion on phentolamine-induced inhibition of the contractile responses of rat thoracic aorta to norepinephrine. The ordinate represents the logarithm of the ratio of the concentration of norepinephrine producing fifty percent of maximum contraction in the presence and absence of phentolamine minus one {Log (dose ratio-1)}. The abscissa represents the log molar concentration of phentolamine. The closed circles represent the Arunlakshana-Schild plot of phentolamine- induced antagonism of norepinephrine in PSS containing 2.5 mM calcium ion. The open circles represent the same experiment performed with

1.6 mM calcium ion in the PSS. The vertical lines represent the standard errors of the same. pA2 ~ is the concentration of antagonist necessary to increase the ED^q of norepinephrine by a factor of 2. The ED5 0 values for norepinephrine were not affected by the increase in the calcium ion concentration in PSS. EFFECT OF CALCIUM ION ON PHENTOLAMINE ANTAGONISM OF NOREPINEPHRINE ON RAT AORTA —•2.5mM CALCIUM N=4 ~ o— o|.6mM CALCIUM N = 6

pA2 - 9 .3 5 ± 0 .3

pA2 -8.21 +0.1

- 1 0 9 8 7 -6 LOG [P H E N TO LA M IN E ] (M O LAR ) 107 postulate was directly tested. The results are summarized in Figures 21 through 23.

The accumulation of 2-C^-serotonin binding to rat thoracic aortae was linear over a period of twenty minutes at 37°C. Saturation of receptor or binding sites appeared to occur after ten minutes as evi denced by the diminution in the slope of the binding curve (Figure 21).

These data are in agreement with the experiments of Thoa, et at, (1969),

Weiss and "Rosecrans (1971 a, b), and Born (1962) employing the guinea- pig vas deferens, ileum, and taenia coli muscle respectively. The initial

slope probably represents the binding of 2-C^-serotonin to both specific receptor sites as well as some non-specific receptor sites which may have

the greatest affinity for this indolealkylamine. The second slope prob

ably represents saturation of an uptake, binding or inactivation mechanism

(Weiss and Rosecrans, 1971 a, b). Therefore, a ten minute equilibration

period was employed in the subsequent studies.

Incubation of thoracic aortae with phentolamine, for thirty minutes

prior to and as well as during, the ten minute equilibration period with

2-C-^-serotonin, inhibited the accumulation of serotonin within the

thoracic aortae (Figure 22). The magnitude of inhibition was related to

the concentration of phentolamine employed in the incubation medium.

Phentolamine (1 X 10“^M) was no more effective an inhibitor of the ac

cumulation of radiolabled serotonin than was 1 0 “% of phentolamine.

However, higher concentrations of phentolamine inhibited further the

accumulation of 2-C^-serotonin binding by the rat thoracic aortae. 108

FIGURE 21

TIME COURSE OF l4 C-SEROTONIN BINDING BY RAT THORACIC AORTA

Accumulation of C^-serotonin in rat thoracic aorta. Each point represents the mean value for four or five aortic strips, each strip representing a single rat. Vertical lines represent the standard error of the mean. The ordinate represents the C^-serotonin in disintegra tions per minute per mg of wet weight of aorta. The abscissa represents • the period of incubation. TIME COURSE OF ,4C-SER0T0NIN BINDING BY RAT THORACIC AORTA

2000 - * p<0.05 t 1600-

1200-

800-

£ 400-

0 4 8 1 2 16 20

TIME (MINUTES) 109 1 1 0

FIGURE 22

EFFECT OF PHENTOLAMINE ON RAT AORTA BINDING OF 1 4 C-5-HT

Effect of phentolamine on the accumulation of C^-4-serotonin by rat thoracic aorta. The ordinate represents the accumulation of radiolabeled serotonin (expressed as disintegrations per minute per mg of wet weight of aorta) by the rat thoracic aorta during a ten minute incuba tion period. The abscissa represents the- concentration of phentolamine.

Vertical lines are the standard error of the mean. Each mean is the average from four or five aortic strips each obtained from an individual rat. An asterisk denotes that the accumulation of C^-4-serotonin in the presence and absence of phentolamine significantly differ from each other. Aortic strips were incubated with phentolamine for thirty minutes before and throughout the ten minute incubation period with

Cl4_serotonin. C-SEROTONIN BINDING (DPM/mg AORTA) 1400 1000 1200 0 0 4 600 800 200 N A ARA IDN O ,4C-5-HT OF BINDING AORTA RAT ON FET F PHENTOLAMINE OF EFFECT (4) O 8 O7 O6 IO"5 IO"6 IO"7 IO-8 (5) HNOAIE (M) PHENTOLAMINE (5) 5 (4) (5) h - * p<0.05 *

1 12

These findings are similar to those observed with phentolamine mediated inhibition of the contractile responses of rat thoracic aortae to serotonin (Figure 9).

The effect of the other autonomic antagonists on the accumulation of 2-C^-serotonin within rat thoracic aortae was examined. The data are summarized in Figure 23. .Cocaine, which enhanced the contractile response to serotonin, diminished the accumulation of the radiolabeled serotonin by the muscle strip. This is similar to cocaine mediated inhibition of the uptake of norepinephrine within adrenergically innervated smooth muscle preparations (Iverson, 1967). Tolazoline did not significantly affect the accumulation of 2-C-^-serotonin within the rat thoracic aortae. Norepinephrine produced a small but significant inhibition of the accumulation of 2 -cl^-serotonin within the thoracic aortae. This effect was not concentration related. It is possible that the small difference between the accumulation of the isotope in thoracic aortae incubated with the two concentrations of norepinephrine represent displacement of serotonin from specific alpha receptors located within or on the cell membrane. Ruffulo, et al, (1976a,b) have demonstrated that only picomoles of norepinephrine must be displaced from specific receptor sites to inhibit the contractile responses to norepinephrine.

This postulate may be supported by the data obtained with cocaine and phentolamine. In thoracic aortae incubated with cocaine, phentolamine did not affect 2-C^-serotonin binding (Figure 23). Similar to that observed with norepinephrine, the change in binding produced by 113

FIGURE 23

EFFECT OF AUTONOMIC AGENTS ON 1 /‘C-5-HT UPTAKE BY RAT THORACIC AORTA

Effect of autonomic agents on the accumulation of C^^-serotonin by rat thoracic aorta. The ordinate represents the accumulation of radiolabeled serotonin by aortic strips over a ten minute incubation period.

The abscissa represents the autonomic intervention employed. The accum ulation of C-^-serotonin is expressed as disintegrations per minute per mg of wet weight of aorta. Vertical lines are the standard errors of the means. An asterisk denotes that the ten minute accumulation of

C^-serotonin in the presence of the autonomic intervention differs significantly (P<0.05) from that of control strips. The numbers in parentheses indicate the number of vascular strips employed in the cal

culation of the mean values. Each vascular strip was obtained from a

separate animal. Antagonists were incubated with the muscle strips for

thirty minutes prior to and throughout the ten minute incubation period with the C^-serotonin. I4C-SER0T0NIN BINDING (DPM/mg AORTA) 1400- 0 0 0 1 1200 1600- - 0 0 4 - 0 0 6 0 0 8 00 2 0

- - - -

OTO COCAINECONTROL NOREPINEPHRINE PRISCOLINE 4 5 (5) (5) (4) ON 0'M xO5 II"M IxIO"5M IxI0"7M IxIO"5M '6M I0 x 5 i C5HT PAE Y RAT THORACIC BY AORTA UPTAKE T 4C-5-H FETO AUTONOMICOF AGENTS EFFECT (5) (5)

COCAINE+PHENTOLAMINE x07 II '5M IxIO IxI0"7M p<0.05 *

115 increasing the concentration of phentolamine from 1 X 10“^ to 1 X 10“5 was negligible. However, it is possible that the specific receptor binding is masked within the mass of nonspecific receptor binding.

Nevertheless, the data clearly demonstrate that phentolamine, in con centrations which inhibit the contractile responses to serotonin inhibit the accumulation of radiolabeled serotonin. Tolazoline neither inhibits the contractile response nor the binding of serotonin to rat thoracic aortae.

The studies of serotonin-mediated contraction of the rat vascular smooth muscle strongly suggested that serotonin-mediated contraction was elicited through alpha receptor stimulation. The alpha receptor seemed to differ from that of the classic alpha receptor since it was sensitive to blockade by phentolamine but not by concentrations of tolazoline which inhibited the contractile responses to norepinephrine. The critical experiment to evaluate this postulate did not provide conclu sive data to refute or verify the postulate that the receptors were different. Although the data seemed to suggest this possibility (Table 4) the relative instability of the rat thoracic aortae during the experi mental period precluded a definitive evaluation of the postulate.

Therefore a more stable vascular smooth muscle preparation was sought with characteristics similar to that observed in thoracic aortae and mesenteric arteries. TABLE 4

SUMMARY OF RAT VASCULAR SMOOTH MUSCLE STUDIES 117

TABLE 4. SUMMARY OF RAT VASCULAR SMOOTH MUSCLE STUDIES

1. The contractile responses of the rat thoracic aortae and mesenteric arteries to serotonin is inhibited by the specific alpha receptor blocking agent phentolamine, but not by tolazoline, a purported non-selective adrenoceptor antagonist.

2. Serotonergic receptor blockade by methysergide inhibits the contractile responses to both serotonin and norepinephrine.

3. These effects are not apparently due to release of vasoactive amines by either agonist since the effects occur despite amine depletion and blockade of the amine re-uptake mechanism located within the adrenergic nerves.

4. Phentolamine, but not tolazoline, inhibits the accumulation of Cl4-serotonin by rat thoracic aortae. This effect is related to the concentration of phentolamine and parallels the inhibitory effect of phentolamine on serotonin-mediated contraction.

5. Phentolamine may interfere with receptor as well as non-receptor binding sites for serotonin.

6 . Serotonin-mediated contraction of rat thoracic aorta and mesenteric arteries may occur by stimulation of alpha receptors or by stimulation of a receptor so similar to the alpha receptor that phentolamine cannot distinguish these receptors.

7. Alternatively, a component of the alpha receptor protein may be an integral moiety of the serotonergic receptor.

8 . The finding that tolazoline, but not phentolamine, can distinguish between the receptor proteins for serotonin and norepinephrine would indirectly suggest the possible existence of two subclasses of alpha adrenergic receptor.

9. The instability of the rat vascular smooth muscle during prolonged incubation with high concentrations- of tolazoline, a prerequisite for characterizing this putative receptor, makes difficult further characterization of the receptors within these preparations.

10. Tolazoline is a more specific antagonist of norepinephrine than is phentolamine. Studies using phentolamine as the specific alpha blocker may possibly have to be re-evaluated. 118

Vanhoutte and co-workers (1969) speculated that the serotonergic receptor within the canine saphenous vein may not be a typical seroto nergic receptor. These investigators suggested that serotonin may elicit venoconstriction in the saphenous vein through stimulation of alpha receptors. Therefore the canine saphenous vein was employed as the second vascular smooth muscle model for a "serotonergic" receptor.

The following studies will clearly demonstrate the serotonergic mediated venoconstriction is via alpha receptor stimulation. Further more the data support the concept that two subclasses of the alpha receptor exist with the vascular smooth muscle cell.

The effect of incubation of canine saphenous veins with norepi nephrine and serotonin on contractile force development is shown in

Figure 24 (left panel). Both norepinephrine and serotonin contracted the saphenous vein. Serotonin was a more potent constrictor of the veins than was norepinephrine, as reflected in the ED^q 's of the agonists

(Table 5). However, the maximal contractile force of the veins was greater when norepinephrine was employed as the agonist than when seroto nin was the agonist (Table 5). The effects of inhibition of the adrenergic neuronal reuptake mechanism for norepinephrine, on the con

tractile responses to serotonin, was investigated. Thirty minutes after

the addition of cocaine to the muscle bath, the sensitivity of the veins

to both norepinephrine and serotonin was enhanced (Figure 24, right panel).

The sensitivity to norepinephrine was enhanced to a greater extent than

the responses to serotonin (Table 5). The maximal contractile responses 119

FIGURE 24

SENSITIVITY OF CANINE SAPHENOUS VEINS TO SEROTONIN AND NOREPINEPHRINE

A comparison of the sensitivity of canine lateral saphenous veins to serotonin and norepinephrine. The ordinate represents the contrac tile force of the vein strips to the agonists, expressed as sensitivity

(percent of the maximum response of the agonist). The abscissa is the molar concentration of agonist. The left panel represents the responses to each of the agonists in control vein strips whereas the right panel represents the responses to each of the agonists in canine saphenous veins incubated throughout the experiment with cocaine (2 X 10“% ) . The vertical lines are the standard errors of the means (S.E.M.). Each value reflects the mean ± S.E.M. from six canine veins which represent six dogs. An asterisk indicates that the response due to norepinephrine differ significantly (P<0.05) from those due to 5-HT. SENSITIVITY (% OF MAXIMUM) lOO-i - 0 4 20 - 0 8 - 0 6 - Q' I~ I I- I0-5 I0-6 7 ” I0 I0~8 '9 IQ HT( ) (6 T -H 5 ESTVT O CNN SPEOU VEI TO S IN E V US SAPHENO CANINE OF SENSITIVITY OTO COCAINE CONTROL EOOI AD OREI P NE IN R EPH EPIN R NO AND SEROTONIN OA CNETAIN F AGONIST OF CONCENTRATION MOLAR P<0.05 * NE (6) IOO-i - 0 4 20 - 0 6 80- - l0 -9 -9 l0 5 -H T (6) T -H 5 iq 8 07 06 I0"5 10"6 |0-7 -8 P<0.05 * E( ) (6 NE

120 TABLE 5

•EFFECT OF NEURONAL REUPTAKE BLOCKADE ON THE CONTRACTILE RESPONSES OF CANINE SAPHENOUS VEINS TO SEROTONIN AND NOREPINEPHRINE 1 22

TABLE 5

EFFECT OF NEURONAL REUPTAKE BLOCKADE ON THE CONTRACTILE RESPONSES OF CANINE SAPHENOUS . VEINS TO SEROTONIN AND NOREPINEPHRINE

PARAMETER N CONTROL COCAINE (5 X 10"%)

ED5 Q (10~7 moles/liter)

Norepinephrine 6 4.74 ± 1.02 0.38 ± 0.08**

Serotonin 6 0.54 ± 0.04* 0.30 ± 0.04**

Contractile force (grams)

Norepinephrine 6 6.76 ± 0.89 6.56 ± 0.62

Serotonin 6 5.33 ± 0.69* 4.32 ± 0.59**

A single asterisk (*) denotes 5-HT responses differ significantly (P<0.05) from responses to NE.

A double asterisk (**) denotes responses of cocaine-treated veins differ significantly (P<0.05) from control responses. 123 of the veins to serotonin was significantly decreased after inhibition of neuronal reuptake of norepinephrine (Table 5). However, the magnitude of norepinephrine-induced contractions of canine saphenous veins was similar before and during inhibition of neuronal reuptake blockade with cocaine (Table 5).

Effect of Phentolamine on the Contractile

Responses of Venous Smooth Muscle

The possibility exists that cocaine-induced enhancement of the sensitivity, as well as inhibition of the maximal contractile response, of the saphenous veins to serotonin was a specific effect resulting from inhibition of serotonin mediated uptake into adrenergic nerves and release of norepinephrine. This possibility was tested in two types of experiments. In the first series of experiments, the ability of phentol amine, the most specific alpha adrenoceptor antagonist currently available, was employed to inhibit the responses to serotonin. In the second series of experiments, the release of 7-H3-norepinephrine was directly measured.

Figure 25 summarizes the results obtained when phentolamine was used to inhibit the contractile responses of canine saphenous veins to nor epinephrine and serotonin. The contractile responses of the veins to norepinephrine were inhibited in a concentration-dependent, competitive manner thirty minutes after the addition of increasing concentrations of phentolamine to the muscle bath. In contrast to these findings, phentolamine was a weak antagonist of the contractile responses of the 124

FIGURE 25

EFFECT OF PHENTOLAMINE ON CONTRACTILE RESPONSES OF CANINE SAPHENOUS VEIN TO NOREPINEPHRINE AND SEROTONIN

Effect of phentolamine on the contractile responses of canine saphenous veins to norepinephrine (panel A) and serotonin (panel B).

The ordinate represents the contractile force generated by the canine vein strips when challenged with the agonists. The abscissa represents

the molar concentration of agonist. The vertical lines represent the

standard error of the mean. C.V. is the coefficient of variation of the experiment. Each mean value represents the responses of six canine veins representing six dogs. The agonists were allowed to remain in

contact with the muscle strips until the contractile responses had

reached equilibrium. Phentolamine was in contact with the muscle strips

for thirty minutes prior to re-evaluation of the responses to the

agonists. An asterisk denotes the responses of the veins to the agonist

in the presence of antagonist significantly differ (P<0.05) from those

obtained in the absence of antagonist (control responses). CONTRACTILE FORCE (GRAMS) 6 - 4 - 5 8 - 7 - -| C.V. = C.V. 47 3. % .2 3 14.7+ I O_8M PHENTOL M 8 _ IO IX O CONTROL « FET F HNOAIE N CONTRACTILE ON PHENTOLAMINE OF EFFECT EPNE O CNN SPEOS EN TO VEIN SAPHENOUS CANINE OF RESPONSES ORPNPRN ® SEROTONIN ® REPINEPHRINE NO OEIEHIE N SEROTONIN AND NOREPINEPHRINE O MLR OCNRTO O AGONIST OF CONCENTRATION MOLAR LOG NE IN M A - 4 - 3 2 - 7 8 6 - 5 - -| - 05 .0 0 < P * 08 I0-7 I0-8

N =6 C.V.= . 2. % .1 2 t 9.3 125 126 vein to serotonin. A small, significant inhibition of the contractile responses to serotonin was observed with 1 X 10"7m phentolamine, whereas a significant inhibition of the contractile responses to serotonin neces sitated a relatively high concentration of phentolamine (1 X 10“^M).

Nevertheless, this concentration of the alpha adrenoceptor blocking agent produced a parallel shift of the concentration response curve to serotonin to the right, suggestive of competitive blockade.

Serotonin may release norepinephrine. Phentolamine-mediated inhibi tion of serotonin-induced venoconstriction could result from blockade of the released norepinephrine acting on the alpha receptors of the veins.

Were this postulate correct then phentolamine should not inhibit the contractile responses to serotonin during inhibition of the neuronal reuptake mechanism with cocaine. This postulate was tested. In contrast to the anticipated findings, incubation with cocaine inhibited the contractile responses to serotonin in venous smooth muscle strips.

Phentolamine was a more potent inhibitor of the contractile responses of the veins to both norepinephrine and serotonin (Figure 26). However, the relative blocking potency against the catecholamine and indole-alkyl amine remained the same (see below).

The ability of an alpha adrenoceptor antagonist to inhibit the contractile responses of the canine saphenous veins to serotonin, especially during inhibition of neuronal reuptake of catecholamines with cocaine, suggested that the effect of phentolamine may be secondary to non-specific depression of the venous smooth muscle cell. However, as 127

FIGURE 26

PHENTOLAMINE MEDIATED INHIBITION OF THE CONTRACTILE RESPONSES OF COCAINE-TREATED (2 X 10“% ) CANINE SAPHENOUS VEINS TO NOREPHINEPHRINE AND SEROTONIN

Effect of cocaine (2 X 10~%) on phentolamine mediated inhibition of the contractile responses of canine saphenous veins to norepinephrine and serotonin. The ordinate represents the contractile force generated by the veins when challenged with norepinephrine (left panel) and serotonin (right panel). The abscissa represents the molar concentration of the agonists. C.V. is the coefficient of variation of the experiment.

Each mean value represents the responses from six canine saphenous veins, each obtained from an individual animal, in the absence (control) and presence of varying concentrations of phentolamine. Phentolamine was present in the muscle bath for thirty minutes prior to re-evaluation of the responses of the veins to the agonists. The standard errors of the individual mean responses did not exceed nine percent of the mean responses. An asterisk denotes that the contractile responses to the agonists in the presence and absence of phentolamine significantly dif fer (P<0.05) from each other. PHENTOLAMINE MEDIATED INHIBITION OF THE CONTRACTILE RESPONSES OF COCAINE - TREATED ( 2 X I0 “6 M) CANINE SAPHENOUS VEINS TO NOREPINEPHRINE AND SEROTONIN

NOREPINEPHRINE SEROTONIN B

* CONTROL

I X10"8 M PHENTOLAMINE

5- 5-

N = 6 IX I0 '6 M

4- C.V. = 4- 11.4+ 3.9% N = 6

*P<0.05 3 - C.V. = 14.1+3.4%

2 -

-9 i-8 -7 1-3

LOG MOLAR CONCENTRATION OF AGONIST 129

summarized in Figure 27, the contractile responses to potassium chloride,

a non-specific smooth muscle stimulant, were similar before and during

alpha adrenoceptor blockade with phentolamine. Similarly, phentolamine-

mediated depression of the contractile responses to serotonin may simply

reflect tachyphylaxis of the venous smooth muscle to serotonin, during

the course of the experiment. As summarized in Figure 28, the contrac

tile responses of the canine veins to both norepinephrine and serotonin were reproducible and stable when repeated concentration-effect curves were obtained in the absence of phentolamine. The concentration-effect

curves presented in Figure 28 represent the initial responses to the

agonists and the fifth concentration-effect curve which reflects the

time period represented by the high concentration of phentolamine. The

concentration-effect curves representing the second, third and fourth

repetitions did not significantly differ (P>0.05) from the initial and

final curves (data not shown). Thus it appears unlikely that phentol-

amine-mediated inhibition of the contractile responses to serotonin was

secondary to non-specific depression of the muscle or tachyphylaxis to

serotonin.

The results with potassium ion differ from the findings of Vanhoutte

and co-workers (1975), who demonstrated that phentolamine inhibited the

contractile responses to potassium chloride. The results are similar to

those of the studies of Greenberg, et al, 1975, which demonstrated that in

helical strips of dorsal metatarsal veins, the contractile responses to potassium chloride were similar before and after inhibition of alpha receptors wit

jhentolamine. There is no explanation at the present time to exolain the 130

FIGURE 27

EFFECT OF PHENTOLAMINE ON THE CONTRACTILE RESPONSES OF CANINE SAPHENOUS VEINS TO POTASSIUM CHLORIDE

Effect of phentolamine on the contractile responses of canine saphenous veins to potassium chloride (50 mM). The ordinate represents the contractile force generated by canine saphenous veins when chal lenged with 50 mM KC1. The abscissa represents the molar concentration of phentolamine. The vertical lines represent the standard error of the mean. Each mean value represents the responses from seven canine saphenous veins each obtained from an individual dog. Note the absence of any significant non-specific depression of the contractile or mem brane properties of the muscle which is believed to be reflected by the responses to this non-specific smooth muscle stimulant. CONTRACTILE RESPONSE TO 50 mM KCI (GRAMS) CHLORIDE POTASSIUM TO VEINS SAPHENOUS CANINE OF RESPONSES - 4 6 3- 5- 2 - - T 0 FET F HNOAIE N H CONTRACTILE THE ON PHENTOLAMINE OF EFFECT >P0.05 =N 7 ~fh 1 “ 08 07 I0"6 I0*7 10*8 ------PHENTOLAMINE ( MOLES / LITER) / MOLES ( PHENTOLAMINE 1 ------' ----

10'

132

FIGURE 28

TEMPORAL RESPONSIVENESS OF CANINE SAPHENOUS VEINS TO NOREPINEPHRINE AND SEROTONIN

Reproducibility and stability of canine saphenous vein responses to norepinephrine and serotonin following repeated concentration re sponse curves. The ordinate represents the contractile force of the canine saphenous vein in response to norepinephrine (panel A) and serotonin (panel B) expressed as a percentage of the maximum response to the agonists. The abscissa represents the molar concentration of the agonist. The solid circles represent the initial concentration response curve obtained for each agonist. The open circles (-0-) repre sent the fifth concentration response curve to the agonist. These were obtained within five hours of the initial responses. The concentratioiv response curves between the initial and final curves were similar

(P>0.05) to the initial and final curves. The vertical lines represent the standard error of the mean. Each value represents the mean response

± S.E.M. from four vein strips, each representing an individual animal. CONTRACTILE FORCE (PERCENT) lOO-i 0 2 - 0 4 - 0 6 - 0 8 - . OEIEHIE . SEROTONIN B. NOREPINEPHRINE A. « —-O FNL EPNE O RESPONSES FINAL O - — - o 09 08 07 06 0'5 I0 I0“6 I0"7 I0"8 I0*9 EPRL EPNIEES F AIE AHNU VEINS SAPHENOUS CANINE OF RESPONSIVENESS TEMPORAL JH. m NTA RSOSS • RESPONSES INITIAL O OEIEHIE N SEROTONIN AND NOREPINEPHRINE TO OA CNETAIN F AGONIST OF CONCENTRATION MOLAR P 5 > .0 0 IOO- 0 2 - 0 4 - 0 8 “ 0 6 - i ------1-9 NTA RESPONSES INITIAL • FNL RESPONSES FINAL O -8 P > 0.05

134 discrepancy between these findings. It is possible that the calcium concentration of the different solutions may play a role in the ability of potassium ion to release norepinephrine (Curro, et al, 1976, see section on phentolamine and calcium ion) and the ability of phentolamine to block this component of the response. Nevertheless the potassium study and the stability study demonstrate that the contractile responses to serotonin and the inherent muscle contractile properties are stable throughout, the experimental procedures employed in this study.

Effect of Serotonin on 7-^H-Norepinephrine

Efflux

Inhibition of neuronal reuptake of norepinephrine enhances the sensitivity of the canine saphenous vein to serotonin. Alpha receptor blockade with phentolamine and inhibition of neuronal reuptake of nor- epiphrine inhibit the force generating ability of serotonin. Phentol amine inhibited the contractile responses to serotonin despite inhibition of the neuronal reuptake mechanism with cocaine. These findings suggest

that serotonin may be taken up into cocaine resistant, as well as sensi

tive adrenergic nerves. Serotonin may then release norepinephrine, the

action of which is then blocked by phentolamine. Alternatively, seroto nin may interact with alpha adrenergic receptors on the venous smooth muscle cell membrane. The data to be presented do not support the former

argument.

Figure 29 summarizes the effect of serotonin on the force develop

ment and release of norepinephrine from adrenergic nerve terminals 135 within the canine venous muscle wall. Superfusion of saphenous vein

strips with serotonin resulted in an increase in active force. The

addition of phentolamine to vein strips contracted by serotonin resulted

in a prompt, irreversible relaxation of the veins. Removal of serotonin

from the superfusate resulted in a return of force to, or below, control

values (Figure 29, top panel). Superfusion of saphenous vein strips with

serotonin did not increase the efflux of 7-H^-norepinephrine from the

adrenergic nerves innervating the venous smooth muscle (Figure 29, bottom

panel). Similar findings were obtained when cocaine was present in the

superfusate (Figure 30). Thus, in the presence and absence of adrenergic

neuronal blockade, serotonin does not release 7-%-norepinephrine from

adrenergic nerves with the smooth muscle of the saphenous vein. However,

phentolamine inhibits the contractile responses to serotonin.

Serotonin is a substrate for monoamine oxidase and the 0-methyl

transferase which may inactivate norepinephrine within the adrenergic

neuron and vascular smooth muscle cell, respectively (Sheppard and

Vanhoutte, 1975; Jarrott, et at, 1975; Berkowitz, et at, 1973). There

fore, for a specific quanta of radioactive material to be released from

the adrenergic nerves during superfusion with serotonin, a dispropor

tionate amount may reflect norepinephrine whereas a lesser amount may

reflect the metabolites. This could result in an increase in norepi

nephrine at the adrenergic neuroeffector junction without any apparent

increase in radioactivity released from the saphenous vein. Since the

previous experiment merely measured total radioactivity, the distribution

of metabolites in the superfusate was evaluated in the presence and

absence of serotonin (Curro, et al, in preparation). 136

FIGURE 29

EFFECT OF 5-HT ON SAPHENOUS VEIN TONE AND 3 H-NE EFFLUX

Changes In force development (top panel) and efflux of 7-3 H- norepinephrine (bottom panel) from canine saphenous veins produced by serotonin and phentolamine. For details of method see text. The vertical lines represent the standard error of the means. The mean values represent the responses of five saphenous veins representing five dogs. An asterisk denotes the force responses of the veins to serotonin in the presence or absence of phentolamine (1 X 10“% ) significantly differ from control force. Serotonin (1 X 10“^M) elicits venoconstric tion which is inhibited by phentolamine despite the absence of any significant increase in the efflux of 7-3 H-norepinephrine. (O z -NE EFFLUX (DPMx I0 3 /M IN ) CONTRACTILE FORCE (G M )± S .E .M . lO.O-i 4.0 5.0- - .0 9 3.0-1 8 2 1.5- 1.0 . . 0 0 - - CONTROL FETO 5H O SPEOS EN TONE SAPHENOUS VEIN ON 5-HT EFFECTOF < ------N H-E EFFLUX H3-NE AND EOOI I SEROTONIN I MI ) S E T U IN (M E TIM xl -> M 6 - lO lx - e PHENTOL AMINE x O M 7 IO’ ------> CONTROL

137 138

FIGURE 30

EFFECT OF 5-HT ON SAPHENOUS VEIN TONE AND 3H-NE EFFLUX AFTER NEURONAL REUPTAKE INHIBITION WITH COCAINE (1 X 10"%)

Effect of inhibition of neuronal reuptake of norepinephrine with cocaine (1 X 10"^M) on the changes in force development and efflux of

7-3H-norepinephrine from canine saphenous veins produced by serotonin and phentolamine. For details of method see text. Cocaine was present in the superfusate throughout the entire experiment. The vertical lines represent the standard error of the means. The mean values represent the responses of five saphenous veins representing five dogs.

An asterisk denotes the force responses of the veins to serotonin in the presence or absence of phentolamine (1 X 10~^M) significantly differ from control force. Serotonin (1 X lCT^M) elicits venocontric- tion which is inhibited by phentolamine despite the absence of any significant increase in the efflux of 7-3H-norepinephrine. n X -N E - EFFLUX (OPM X I0 5 ) ± S.E.M. CONTRACTILE FORCE (GM) t S.E.M. 10 3.0-1 6 2 0- .0 9 8 . - 4.0 5.0- 7.0- l. . . . .0-1 0 0 0 0 J - - - CONTROL - FLX FE NUOA REUPTAKE NEURONAL AFTER EFFLUX E 5-N H D N A FET F - N AHNU VI TONE VEIN SAPHENOUS ON T 5-H OF EFFECT NIIIN IH OAN (XI _6M) IO (IX COCAINE WITH INHIBITION 8 6 0 38 4 3 30 26 2 2 18 EOOI I 0"7 " I0 IX '6M IO SEROTONIN IX COCAINE IE MI ) S E T U IN M ( TIME PHENTOLAMIN I X I0~6 M I0~6 X I

2 72 0 7 6 6 62 8 CONTROL

139 140

It was found that serotonin did not affect the metabolism of

7-3H -norepinephrine with the untreated or cocaine treated saphenous vein. The addition of phentolamine to venous smooth muscle contracted by serotonin, did not significantly affect the metabolism of norepinephrine. These findings (data not shown) suggest that phentol amine may exhibit cross-sensitivity with the serotonergic receptor or that serotonin may interact with alpha adrenergic receptors in the saphenous vein.

Effect of Methysergide on Canine Venous Responses to Serotonin and Norepinephrine

The results of the preceding experiments suggested that phentol amine inhibits the contractile responses of canine veins to serotonin either by interacting with the serotonergic receptor or the alpha adrenergic receptor. Methysergide is a potent antagonist of serotonin- induced contractions in many biologic systems (Goodman and Gilman, 1975).

Blockade of serotonergic receptors with methysergide should ablate the responses of the veins to serotonin with minimal inhibitory effects on the contractile responses of the veins to norepinephrine. Thus the 141 characteristics of the serotonergic receptor of canine saphenous veins was examined. Methysergide, in concentrations up to 10“^M did not inhibit the contractile responses of canine saphenous veins to either norepi nephrine or serotonin. Higher concentrations of methysergide, could not be studied since it elicited venoconstriction (Figure 31). The venoconstrictor response to methysergide was abolished by phentolamine.

Therefore, the effect of methysergide was re-evaluated in vascular strips incubated with phentolamine and tolazoline, two alpha adrenoceptor antagonists. The results are summarized in Figures 32 and 33. Despite relatively complete alpha adrenergic receptor blockade, in the presence and absence of adrenergic neuronal reuptake mechanism, methysergide was not an antagonist of serotonergic mediated venoconstriction. These findings suggest that the "serotonergic receptor" in canine saphenous veins are different from the "classical" serotonergic receptors in non- vascular smooth muscle (Innes and Kholi, 1968).

Effect of Alpha Adrenoceptor Antagonists on

Serotonin-Induced Venoconstriction

In an attempt to evaluate the nature of the serotonergic receptor in canine saphenous veins, the characteristics of phentolamine-induced blockade was examined in more detail. Figure 34 is an Arunlakshana-

Schild plot of phentolamine-mediated inhibition of serotonin and norepinephrine-induced contraction. The slopes of the Log (dose ratio-1) versus Log molar concentration of phentolamine are not significantly 142

FIGURE 31

EFFECT OF METHYSERGIDE ON THE RESPONSES TO SEROTONIN AND NOREPINEPHRINE IN CANINE SAPHENOUS VEIN

Effect of serotonergic receptor blockade by methysergide on canine saphenous vein responses to norepinephrine and serotonin. The ordinate represents the contractile responses of saphenous veins expressed as a percentage of maximum response to each agonist. The abscissa represents the molar concentration of the agonist. The vertical lines are the standard errors of the means. Each value is the mean of four saphenous veins. An asterisk denotes the concentration response curves in the presence of methysergide differ from responses in the absence of methy

sergide (P<0.05). Note the absence of inhibition of the responses to norepinephrine and serotonin. Higher concentrations of methysergide

could not be employed due to contraction of the saphenous vein. CONTRACTILE RESPONSE (% MAXIMUM ± S E M ) 0 0 1 - 0 3 - 0 4 H 0 9 20 - 0 7 1— • CONTROL • 110—| - 0 6 50- - 0 8 0 1 0 IHR OCNRTOS F S CUE A HNE N ISOI TNIN OF TENSION DIASTOLIC IN CHANGE A CAUSED MSG OF CONCENTRATIONS HIGHER * * - - - - 05 DE NT IFR INFCNL FO CONTROL FROM SIGNIFICANTLY DIFFER NOT DOES 5 .0 0 > P * ACLR STRIPS VASCULAR A a O1 I‘9 0* I O'7 *8 I0 9 I0‘ IO-10

* * 7 ' 0 I METHYSERGIDE '8 0 1 N A - EOOI ( LI ) R E IT /L S E L O (M SEROTONIN

S I 7 16±0. X 0' * '8 I0 X .7 0 ± 1.6 '7M IO MSG OTO 33 8 O'8 IO X .8 0 3.3± CONTROL E ONI NEPHRI N NE SAPHE IN E V S U O EN H P A S E IN N A C IN E IN R H P E IN P E R O N D N A IN N TO O SER

F DE O THE RE ES TO S SE N O P ES R E H T ON E ID G R E S Y H T E M OF T C E F F E ED S0 to*8

0'5 I0 + + o o z I- 20- S ce < _l LU 4°- £ V) o z 60- a if) OL Ui 1

- 0 8 B 0 -

OEIEHIE MOLSLTR) LES/LITER O (M NOREPINEPHRINE

1' I0' I s 0' 3 I '4 XIO 3 '4 I0 's I0 '8 0 I 10'7 8 ' 0 I S I0' 21 5 I" * XIO"6 .5 0 ± 2.1 M '7 0 I MSG OTO 37 02XI "8 IO X 0.2 3.7± CONTROL ed 50

143 144

FIGURE 32

EFFECT OF METHYSERGIDE ON THE RESPONSES TO NOREPINEPHRINE IN THE PRESENCE OF PHENTOLAMINE AND TOLAZOLINE

Effect of serotonergic receptor blockade by methysergide on canine saphenous vein responses to norepinephrine, during alpha receptor blockade with phentolamine (1 X 10“% ) , in combination with tolazoline

(1 X 10“% ) in the presence and absence (control) of cocaine hydro chloride (2 X 10“^M) an inhibitor of the adrenergic neuronal reuptake mechanism within the adrenergic nerve terminal. The ordinate represents the contractile responses of saphenous veins expressed as a percentage of maximum response to each agonist. The abscissa represents the molar concentration of the agonist. The vertical lines are the standard errors of the means. Each value is the mean of four saphenous veins.

An asterisk denotes the concentration response curves in the presence of methysergide differ from responses in the absence of methysergide

(P>0.05). CONTRACTILE RESPONSES (% MAXIMUM t S E M ) - 0 4 - - 80- - - 0 6 - - 0 0 1 140- 120 40- 40- - 0 6 - 0 8 20 20 0-1 - - - - -

OEIEHIE(MOLSLTR) LES/LITER O M ( NOREPINEPHRINE 0' 3I" I 5 0_S I0 X 3 *5 I0 3XI0"6 '6 I0 '6 0 I I0-8 N =N 4 MSG CONTROL EFFECT OF METHYSERGIDE ON THE RESPONSES TO RESPONSES ON THE OF METHYSERGIDE EFFECT CONTROL NOREPINEPHRINE IN THE PRESENCE OF INPRESENCE THE NOREPINEPHRINE PHENTOLAMINE AND AND TOLAZOLINE PHENTOLAMINE o CO CL £ to UJ 2 0 0 1 140- 120 160- 180- - 0 4 20 - 0 8 60- - - - OEIEHIE MOL LT ) R /LITE S LE O (M NOREPINEPHRINE P<0.05 *

N =4

OAN 2XO6 M XIO-6 2 COCAINE M Ul 146

FIGURE 33

EFFECT OF METHYSERGIDE ON THE RESPONSES TO SEROTONIN IN THE PRESENCE OF PHENTOLAMINE AND TOLAZOLINE

Effect of serotonergic receptor blockade by methysergide on canine

saphenous vein responses to serotonin, during alpha receptor blockade with phentolamine (1 X 10~^M), in combination with tolazoline (1 X 10”% )

in the presence and absence (control) of cocaine hydrochloride

(2 X 10“^M) an inhibitor of the adrenergic neuronal reuptake mechanism within the adrenergic nerve terminal. The ordinate represents the

contractile responses of saphenous veins expressed as a percentage of

maximum response to each agonist. The abscissa represents the molar

concentration of the agonist. The vertical lines are the standard errors

of the means. Each value is the mean of four saphenous veins. An

asterisk denotes the concentration response curves in the presence of

methysergide differ from responses in the absence of methysergide

(P<0.05). CONTRACTILE RESPONSES (% MAXIMUM 1 SEM) - 0 4 - - 0 8 - - 0 6 - - 0 0 1 120-1 - 0 4 - 0 8 20 - 0 6 20 - - - ICT7 8 - 0 I 9 ‘ 0 I A|0'8 ■ ■ I CONTROL • E ONN ( LI ) R E IT /L S E L O (M NIN TO O SER =4 = N G S M 0 ‘® CONTROL EFFECT OF METHYSERGIDE ON THE RESPONSES TO RESPONSES ON THE OF METHYSERGIDE EFFECT

SEROTONIN IN THE PRESENCE OF IN PRESENCE THE SEROTONIN PHENTOLAMINE AND TOLAZOLINE AND PHENTOLAMINE +i 2 CO J U S o o z - J J U O C L C o z O C UJ < x 5 3 QC < o a: vP 2 40n 0 I4 0 0 1 120 - 0 4 20 - 0 6 - 0 8 - - -

0‘9 0’8 0' I 6 I -5 I0 -6 I0 '7 I0 8 ’ I0 9 ‘ I0 EOOI ( LI ) R E IT /L S E L O (M SEROTONIN T

4 = N OAN 2XI0'6 M 6 ' 0 I X 2 COCAINE

m 148

FIGURE 34

EFFECT OF PHENTOLAMINE ON THE CONTRACTILE RESPONSES OF CANINE SAPHENOUS VEINS TO NOREPINEPHRINE AND SEROTONIN

Effect of phentolamine on the contractile responses of canine saphenous veins to norepinephrine and serotonin. The ordinate repre sents the logarithm of the ratio of the concentration of agonist producing 50 percent of maximum contractile response in the presence and absence of antagonist minus one (dr-1). The abscissa represents the log molar concentration of the antagonist. The intercept of the line with the abscissa represents the concentration of antagonist necessary to increase the ED5 Q of the agonist by 1 0 0 percent, i.e., a shift to the right by a factor of 2 (pA2 )• The lines represent the mean responses

± S.E.M. The stippled curves represent the values obtained in vascular strips incubated with cocaine HC1 (2 X 10“% ) for three hours prior to and throughout the experiment. Each value represents the mean responses from six canine saphenous veins representing six dogs. LOG ( DOSE RATIO - I ) -lJ 3-i AHNU VIS O NOREPINEPHRINE TO VEINS SAPHENOUS pA2 OTATL RESPONSESCONTRACTILE CANINE OF -8 FETO PETLMN ON THE PHENTOLAMINEEFFECT OF O [ OL NE] MOLAR) O (M ] E IN M LA TO N E H [P LOG CONTROL N = 6 N SEROTONINAND 7 -6 - -7 OREI P NE IN R EPH EPIN R NO PA 2

EOT NIN TO SERO

5 - 149 150

different from the theoretical value of 1 , do not deviate from paral lelism, and intersect the abscissa at different concentrations of phentolamine. These data suggest that phentolamine inhibits norepineph rine and serotonin at the same or similar receptors. However, phentol amine is a more potent antagonist against norepinephrine than serotonin

(Figure 25, Table 6 ). Furthermore, the ratio of pA£ values against norepinephrine and serotonin remain constant despite adrenergic neuronal blockade by cocaine. These findings suggest that the serotonergic receptor is similar to the alpha adrenergic receptor.

The generality of this concept was tested with another alpha adrenoceptor antagonist, tolazoline. In contrast to the findings with phentolamine, tolazoline was a less potent antagonist of norepinephrine-

induced venoconstriction, than was phentolamine (Figure 35 , left panel;

Table 6 ). Tolazoline produced a competitive blockade of the responses

to norepinephrine. However tolazoline did not inhibit, but rather

enhanced, the contractile responses to serotonin (Figure 36 , left panel).

Similarly, after inhibition of neuronal reuptake of norepinephrine, and

probably serotonin, tolazoline inhibited the responses to norepinephrine

but enhanced the responses to serotonin {Figures 35 and 36 , right

panel(s)}. These findings are inconsistent with the postulate that

norepinephrine and serotonin interact with the same alpha adrenergic

receptor. The ability of tolazoline to inhibit norepinephrine and not

serotonin would suggest that these agonists act on different phentolamine

sensensitive, vide infra alpha adrenergic, receptors. TABLE 6

pA, VALUES OF PHENTOLAMINE AND TOLAZOLINE AGAINST NOREPINEPHRINE AND SEROTONIN ON CANINE SAPHENOUS VEIN 152

TABLE 6 . pA2 VALUES OF PHENTOLAMINE AND TOLAZOLINE AGAINST NOREPINEPHRINE AND SEROTONIN ON CANINE SAPHENOUS VEIN

ANTAGONIST TREATMENT N NE 5-HT (pA2-log moles/Liter) ± S.E.M.

Phentolamine Control 6 7.72 ± 0.08 6.05 ± 0.28

Phentolamine Cocaine 6 8.36 ± 0.13* 6.71 ± 0.17 2 X 1 0 " %

Phentolamine Tolazoline 5.54 ± 0.16* 6.65 ± 0.16 10“%

Phentolamine Cocaine 6.29 ± 0.37 7.38 ± 0.05* 2 X 1 0 " %

Tolazoline 10~ %

Tolazoline Control 4 5.68 ± 0.28 No Inhibition

Tolazoline Cocaine 4 6.19 ± 0.14 No Inhibition 2 X 1 0 - %

pA2-concentration of antagonist necessary to increase the ED5 0 concentration of agonist by a factor of two.

* Responses differ significantly (P<0.05) from corresponding control.

Each value represents the mean ± S.E.M. of the number of dogs (N) from which vascular strips were obtained. 153

FIGURE 35

EFFECT OF TOLAZOLINE ON RESPONSES OF CANINE SAPHENOUS VEIN TO NOREPINEPHRINE

Effect of alpha receptor blockade by tolazoline on canine saphenous vein responses to norepinephrine. The control venous smooth muscle shown on left panel. Vein strips incubated with cocaine to inhibit adrenergic neuronal reuptake (right panel). The ordinate represents the contractile force of the veins elicited by norepinephrine (expressed as a percent of control responses) in the absence (control) and presence of increasing concentrations of tolazoline. The abscissa represents the molar concentration of norepinephrine. Vertical lines are the standard errors of the means. Each value represents the mean response obtained from four saphenous veins representing four dogs. An asterisk denotes a significant (P<0.05) inhibition of the curve from control values. This is in contrast to tolazoline-mediated enhancement of the contractile responses of the veins to serotonin (Figure 38). CONTRACTILE RESPONSES ( %MAXIMUM t SEM)

100- I20-|

- 0 2 - 0 3 in - 0 9 - 0 4 - 0 7 110- 0- 8 60- 10-

- 0 o i OEIEHIE MOL LT ) R /LITE S LE O (M NOREPINEPHRINE 10" - • • A o A * O ■ P<0.05 TOLAZOLINE CONTROL 10 -8 CONTROL AIE AHNU VI T NOREPINEPHRINE TO VEIN SAPHENOUS CANINE 10 FET F OAOIE N EPNE OF RESPONSES ON TOLAZOLINE OF EFFECT -7 10 r 6 I0-5 I ~ o < o to o or UJ a: UJ CO O uj CO 80H I 90 1 2 CL co x 3 IOOH - 0 6 0 3 40H 0 5 20 20 H 70H 110 0 2 0 1 0

OEIEHIE MOLSLTR) LES/LITER O (M NOREPINEPHRINE 10 P<0.05 * 9 - OAN 2 06 M I0"6 2X - COCAINE N= 4

10 ' 10

1-7 0 1 '

I0~5 154 155

FIGURE

EFFECT OF TOLAZOLINE ON RESPONSES OF CANINE SAPHENOUS VEINS TO SEROTONIN

Effect of alpha receptor blockade by tolazoline on canine saphenous vein responses to serotonin. The control venous smooth muscle shown on left panel. Vein strips incubated with cocaine (2 X 10“% ) to inhibit adrenergic neuronal reuptake (right panel). The ordinate represents the contractile force of the veins elicited by serotonin (expressed as a percent of control responses) in the absence (control) and presence of increasing concentrations of tolazoline. The abscissa represents the molar concentration of serotonin. Vertical lines are the standard errors of the means. Each value represents the mean response obtained from four saphenous veins representing four dogs. An asterisk denotes a significant (P<0.05) enhancement of the curves from control values.

This is in contrast to tolazoline-mediated inhibition of the contractile responses of the veins to norepinephrine (Figure 37). CONTRACTILE RESPONSES (% MAXIMUMt SEM) 130- 30- 110 50- 70- 90- 0 1 - - I "7 I0 & I0'8 0 I A CONTROL • P<0.05 * TOLAZOLINE N = 4 O 1 I' I* I’ 10" I0’7 I0*e I0'9 IO‘10

5-H T (MOLES/LITER) T 5-H

AIE AHNU VIS O SEROTONIN TO VEINS SAPHENOUS CANINE FET F OAOIE N EPNE OF RESPONSES ON TOLAZOLINE OF EFFECT CONTROL T 10 -3 o o OC UJ to Q. 50- o to UJ z I30-. N = 4 P<0.05 Q- 0 -1 IQ

COCAINE q i 5-HT (MOLES/LITER) 5-HT Q-8 8 - IQ 9 - q i 7 - q i 6 - ■a * q i

5 - 156 157

Effect of Phentolamine on Serotonin Mediated

Venoconstriction After Alpha Receptor Blockade

With Tolazoline

The results of the preceding experiments suggested the existence of two distinct alpha adrenoceptor antagonists in canine venous smooth muscle. One sensitive to inhibition by tolazoline (tentatively desig nated alpha^) and the second receptor sensitive to inhibition by phentolamine (tentatively designated as a lpt^). It is obvious that phentolamine also acts to inhibit alpha^ (Figures 25 and 26). If this premise is correct, and serotonin elicits its contractile response by

stimulation of alpha2 receptors, whereas norepinephrine may activiate both alpha^ and alpha2 receptors, then inhibition of alpha^ receptors

with tolazoline should result in a diminished activity of phentolamine

against norepinephrine with an enhanced potency against serotonin. The

postulate was tested. Saphenous vein strips incubated with tolazoline

(1 X lO-^M) for three hours demonstrated relatively stable, reproducible

responses to both norepinephrine and serotonin during a four hour

experimental period (Figure 37). In canine saphenous vein strips

incubated with tolazoline, in the presence and absence of cocaine, the

contractile responses to norepinephrine were not affected by phentol

amine until the concentration of this alpha antagonist had reached

1 X 10”% . Further increases in the- concentration of phentolamine

produced a concentration related increase in the inhibition of the

responses to norepinephrine (Figure 38). Phentolamine-mediated inhibi

tion of the contractile responses to serotonin was more pronounced in 158

FIGURE 37

TEMPORAL RESPONSES OF CANINE SAPHENOUS VEIN TO NOREPINEPHRINE AND SEROTONIN IN THE PRESENCE OF 10“% TOLAZOLINE

Stability of canine saphenous vein responses to serotonin and norepinephrine during extended incubation with tolazoline (1 X 10“^M) and cocaine (2 X 10“^M). The ordinate represents the contractile force generated upon addition of serotonin or norepinephrine to the muscle bath (expressed as a percent of maximum control response). The abscissa represents the molar concentration of agonist. Vertical lines are the standard errors of the means. Each value represents the mean response from four vascular strips each representing a single animal. Tolazoline and cocaine were present in the muscle bath during the three hour equilibration period as well as throughout the entire experiment. The final response represents the concentration effect curve for the agonist obtained four hours after the initial concentration effect curve. Curves obtained in between the initial and final curves did not differ from the final concentration response curve. CONTRACTILE RESPONSE ( % MAXIMUM! SEM) loo-, 40- 60- 80- 0 2 A 0 ««* «* - - * IA RESPONSE FINAL IH 0 IUE' QIIRTO BTEN AH OE RESPONSE DOSE BETWEEN EACH EQUILIBRATION MINUTES' 30 WITH EXPERIMENT OFTHE TOLAZOLINE COURSE M THE I0'4 THROUGHOUT PLUS AND TO PRIOR PSS HOURS IN 3 FOR ALLOWED TOEQUILIBRATE WERE STRIPS VASCULAR RESPONSE DOES INITIAL FROM SIGNIFICANTLYNOTDIFFER P>0.05 OEIEHIE (MOLES/LITER) NOREPINEPHRINE Iff 1-8 0 - - - 0 • IA 18 01XO3* XIO'3 +0.1 1.8 FINAL 3.95+0.3X10'3 INITIAL ----- • INITIAL RESPONSE INITIAL • FINAL RESPONSE*** FINAL Iff 1-7 E ORL EPNE O CNN SPEOS VEIN SAPHENOUS CANINE OF RESPONSES RAL PO TEM 10 N H PEEC O I0'4 T AZ I * * E LIN ZO LA TO M 4 ' 0 I OF PRESENCE THE IN -6 ED IS THE O OEIEHIE N SEROTONIN AND NOREPINEPHRINE TO 10 EN E O FU TM CNRL OF CONTROLS EACH VASCULAR(N STRIP = TIME FOUR4) OF SEM + MEAN -3

—I I0'4 3XI0"4 I0'4 -- 1 o o 2 I- cc < o i- _i J U J U cr LU 40- cn Q- O 2 co < S? s - 0 8 x 5 U) 2 l0°-l " > Z

20 60- B 0- -

INITIAL 1.8+ 0.45 1.8+XIO-8 0.45 INITIAL FINAL N= 4 SEROTONIN (MOLES/LITER) .8+ 0 .6 X I0 '8 * '8 I0 X .6 0 .8+ 10'8

I0'6

160

FIGURE 38

EFFECT OF TOLAZOLINE-INDUCED ALPHA ADRENERGIC RECEPTOR BLOCKADE ON THE PHENTOLAMINE- MEDIATED INHIBITION OF NOREPINEPHRINE AND SEROTONIN ON CANINE SAPHENOUS VEIN

Effect of phentolamine on canine saphenous vein responses to norepinephrine and serotonin during alpha receptor blockade with

tolazoline (1 X 10“^M). The ordinate represents the contractile

responses of saphenous veins expressed as a percentage of maximum

response to each agonist. The abscissa represents the molar concentra

tion of the agonist. The vertical lines are the standard errors of the means. Each value is the mean of four saphenous veins. An asterisk

denotes the concentration response curve in the presence of phentolamine

differ from responses in the absence of phentolamine (P<0.05). CONTRACTILE RESPONSE (% MAXIMUM +SEM) 0 0 1 120-1 40- 80- 60- 20 0 O E I E H I E ( O E / I E )SEROTONIN (MOLES/LITER) NOREPINEPHRINE (MOLES/LITER) - - - P<0.05 * 10 T EETR BLOCKADERECEPTOR PHENTOLAMINEMEDIATED - ON THE r I0"8 PHENTOLAMINE CONTROL N N = 4 6 FETF OAOIEIDCD ALPHA TOLAZOLINE-INDUCED ADRENERGIC EFFECTOF 3X10 T INHIBITION SEROTONIN OF AND NOREPINEPHRINE -6 N AIE SAPHENOUSON CANINE .VEIN 10 -5 3X10 1 -5 u o i z t— cc < u o (/) ” UJ d Ul a. UJ

20H 0 0 1 120-1 40- 60- 0 -

0 9 08 07 06 I0-5 I0‘6 I0-7 I0‘8 I0-9 P<0.05

** 162 venous strips incubated with tolazoline (Figure 40). Phentolamine- induced blockade of serotonin mediated venoconstriction was competitive, occured with an initial concentration of 1 X 10”^M phentolamine, and resulted in a thousand fold shift to the right of the concentration effect curve for serotonin. Similar findings were observed in venous smooth muscle strips incubated with tolazoline and cocaine (2 X 10“% ) to inhibit neuronal reuptake of norepinephrine and serotonin (Figure 39).

These experiments support the concept that serotonin and norepinephrine interact with two distinct receptors in the canine saphenous vein.

These receptors appear to be alpha receptors but differ in sensitivity to the adrenoceptor blocking agent, tolazoline. These findings suggest the existence of two subclasses of alpha adrenoceptors in canine venous smooth muscle. Serotonin appears to elicit venoconstriction by stimu lation of alpha2 receptors. 163

FIGURE 39

EFFECT OF TOLAZOLINE-INDUCED ALPHA ADRENERGIC RECEPTOR BLOCKADE ON THE PHENTOLAMINE-MEDIATED INHIBITION OF NOREPINEPHRINE AND SEROTONIN IN CANINE SAPHENOUS VEIN

Effect of alpha adrenergic receptor blockade by phentolamine on canine saphenous vein responses to norepinephrine and serotonin during alpha receptor blockade with tolazoline (1 X 10“^M) and inhibition of adrenergic neuronal reuptake with cocaine hydrochloride (2 X 10"^M).

The ordinate represents the contractile responses of saphenous veins expressed as a percentage of maximum response to each agonist. The abscissa represents the molar concentration of the agonist. The verti cal lines are the standard errors of the means. Each value is the mean of four saphenous veins. An asterisk denotes the concentration response curves in the presence of phentolamine differ from responses in the absence of phentolamine (P<0.05). CONTRACTILE RESPONSE (% MAXI MU M + SEM) 0 0 1 120-1 - 0 4 60- 80- 20 OEIEHIE MOLSLTR) EOOI (MOLES/LITER) SEROTONIN ) LES/LITER O (M NOREPINEPHRINE - ISE WR ALWD O QIIRT FR HE HUS N S CONTAINING PSS IN HOURS THREE FOR EQUILIBRATE TO ALLOWED WERE TISSUES TOLAZOLINE ( I0 '4 M) PLUS COCAINE COCAINE PLUS M) '4 I0 ( TOLAZOLINE - ORE F H EXPERIMENT THE OF COURSE P<0.05 * I0"8 A a I -S I0 o OTO CCIE COCAINE COCAINE I0"6 ■ CONTROL • RECEPTOR BLOCKADE ON THE PHENTOLAMINE - MEDIATED - PHENTOLAMINE THE ON BLOCKADE RECEPTOR I0-7 FET F OL OLNEI D APA ADRENERGIC ALPHA ED C U D E-IN LIN ZO LA TO OF EFFECT PHENTOLAMINE N =4 NIIIN F OEIEHIEAD SEROTONIN AND NOREPINEPHRINE OF INHIBITION

N AIE AHNU VI ' VEIN SAPHENOUS CANINE IN o llJ L co (/>Ui 2XI M) 6 ‘ I0 X (2 l J 120-1 - 0 4 80- 60- 20 - P <0.05 * N = 4 RO T AD HOGOT THE THROUGHOUT AND TO PRIOR

164 TABLE 7

SUMMARY OF CANINE SAPHENOUS VEIN STUDIES 166

TABLE 7. SUMMARY OF CANINE SAPHENOUS VEIN STUDIES

1. The contractile responses of canine saphenous vein to serotonin and norepinephrine are inhibited by the specific alpha receptor blocking agent phentolamine, but not by methysergide, a serotonergic receptor antagonist.

2. A non-selective alpha receptor antagonist, tolazoline, selectively inhibits the contractile responses to norepinephrine but not serotonin.

3. During alpha receptor blockade by tolazoline, phentolamine is a more selective antagonist of the contractile responses to serotonin than to norepinephrine.

4. Serotonin does not significantly increase the release of 7 - % - norepinephrine or alter the metabolism of norepinephrine in the isolated superfused saphenous vein preparation.

5. The selective antagonism of tolazoline, in contrast to that of phentolamine, suggest the existence of two apparently different receptors sensitive to inhibition by phentolamine.

6 . ■ The data suggest the existence of two subclasses of alpha adrenergic receptor in canine saphenous veins. DISCUSSION

Serotonin and Rat Vascular Smooth Muscle

Serotonin and norepinephrine have in common many properties. Both biogenic amines can elicit a contraction of rat thoracic aortae and mesenteric arteries. Both amines have an uptake, storage and release mechanism which display the same ionic dependency and require many of the same cofactors (Goodman and Gilman, 1975). Norepinephrine and serotonin are both degraded by catechol-o-methyl transferase (COMT) and type A monoamine oxidase (MAO) (Jarrott, 1971). The metabolite of

O-methylation represents the amine which has been subjected to extraneuronal uptake, metabolism, and subsequent diffusion from the cell.

The MAO pathway represents the amine metabolized after neuronal uptake.

The amines which have been both deaminated and 3-methoxylated possibly represents the amine leaking from the neuronal storage granules into the nerve cytoplasm (Luchelli— Fortis and Langer, 1975). In addition, sero

tonin and norepinephrine may share the same uptake process involved in

the termination of their action at the effector organ (Snipes, Thoenen and

Tranzer, 1968; Buchanon, et al, 1974; and Potter and Axelrod, 1962).

Serotonin is a potent constrictor of vascular smooth muscle in vivo

and in vitro (Gulati, et al, 1968; Furchgott, 1955; and Freyburger, et al,

1952). Serotonin mediated increases in vascular smooth muscle tone are

167 168 dependent upon many factors. The major determinants of the contractile response to serotonin include: (1 ) the species from which the vascular smooth muscle is obtained; (3) the vascular bed employed within a given species; (3) the state of the vascular smooth muscle contraction; (4) the density and symmetry (adventitial versus medial) of the adrenergic innervation; and (5) the distance from the adrenergic nerve terminal to the smooth muscle cells within the synaptic cleft (Trendelenburg, 1972).

The contractile response to serotonin may be mediated by the release of calcium ion from intracellular or membrane bound calcium pools

(Bloomquist and Curtis, 1972; Greenberg, et al, 1973; Bohr, et al> in press). Serotonin induced vasoconstriction is believed to be mediated

through specific receptor proteins on the vascular smooth muscle cell membrane, i.e., a structurally specific serotonin receptor (Furchgott,

1954, 1955).

Despite the postulate of specific peripheral vascular smooth muscle

receptors for serotonin, the literature is replete with evidence that

serotonin may interact with the adrenergic nervous system. Freyburger,

et alt (1952) observed that phenoxybenzamine, an alpha adrenoceptor

antagonist, inhibited the pressor responses of anesthetized dogs to

serotonin. However, the degree of inhibition was significantly less than

that obtained with norepinephrine or with epinephrine. Furchgott (1954)

subsequently demonstrated that serotonin could protect the alpha

adrenergic receptor of rabbit thoracic aortae from blockade by alkylating

adrenoceptor blocking agents. These findings were subsequently confirmed

by Innes and Kohli (1970). Both groups of investigators concluded that

specific serotonin and adrenergic receptors existed in vascular smooth 169 muscle but that cross-reactivity existed between alpha and serotonergic receptor stimulants. Several sympathomimetic amines can also elicit contraction of the guinea pig ileum by interacting with serotonin receptors (Innes, 1963; Innes and Kohli, 1969). Clement and co-workers

(1969) suggested that the parallelism between serotonin and norepineph rine in canine saphenous veins may indicate that both drugs could act on the same or similar receptor. A similar conclusion was reached by

Offermerer and Ariens (1966) employing the rat vas deferens and by Wakade, et at, (1970) employing the cat nictitating membrane. Despite the sug gestions that serotonin may elicit contractions by a direct interaction with alpha adrenergic receptors, neither a detailed analysis of the interaction nor a definitive solution to the problem has been forth coming.

Evidence is now presented that the serotonergic induced contrac

tion of the rat thoracic aortae and mesenteric artery is mediated by stimulation of alpha-adrenoceptors. Furthermore, this adrenergic receptor (hereafter referred to as ag) is capable of being differen

tiated from the classical noradrenergic alpha-adrenocepter (hereafter

referred to as a^) by the 2 -substituted imidazoline compound, tolazoline

(Figure 40).

For the analysis and characterization of adrenergic receptors, two

procedures are usually employed: (1 ) the establishment of the relative

potencies of a series of adrenergic agonists and (2 ) the evaluation of

the potency of an antagonist for its ability to block or inhibit the FIGURE 40

STRUCTURAL FORMULAS OF SEROTONIN (5-HT), NOREPINEPHRINE (NE) AND ANTAGONISTS 171

STRUCTURAL FORMULAS OF 5-HT, NE AND ANTAGONISTS

Hi H i H/ C-C-H C-C-N i i \ H NH OHH H HO NOREPINEPHRINE 5 - HYDROXYTRYPTAMIN E (5-H T)

H CH, / SC — f ,N> A • H - C - f > H HN- /T\ H HN 1 TOLAZOLINE

HO' PHENTOLAMINE CH-: H CH2 -CH3 « ^-CO-O/JHCHgCH-N7 0 *_ i. i i.. J*. / CH2CHfCH CH(C00CH3)-

COCAINE N-CH

METHYSERGIDE response to a given agonist. If one or both of these methods is to be followed to differentiate subclasses of receptors within a specific class of receptors, strict control of the experimental conditions is absolutely critical (Furchgott, 1972). The experimental criteria established by Furchgott (1964, 1972) were followed to achieve the optimal conditions for the pharmacological characterization of the alpha adrenoceptors in the rat thoracic aortae, rat mesenteric artery and in the canine saphenous vein. These conditions stated briefly should include: proper time for the competitive antagonist to achieve equilib rium in the region of the receptors; awareness in any change in sensitivi ty of the preparation during the course of the experiment; the response of the tissue preparation to an agonist should be due solely to the direct action of the agonist on one type of receptor; measurement of the response to a given dose of agonist should be at the maximal level achieved by the tissue preparation and proper time controls should be incorporated in the experimental design. In experiments used to deter mine whether a receptor responsible for a given response should be placed in the a- or 2 - class, conditions usually need not be controlled very rigorously (Furchgott, 1972).

The basic assumption in the experimental design is that similarities or differences in the characteristics of responses reflect similarities or differences in the characteristics of the receptors mediating these responses. Thus, if two different responses are obtained by a similar series of agonists, and the potency for an antagonist is similar against the agonists, then the interpretation is 173 that the two responses are mediated by the same type of receptor.

Alternatively, a finding of dissimilar relative potencies or of dis similar potencies for the antagonist, or of both, would indicate the responses to the two agonists are mediated by different types of receptors.

The alpha-adrenoceptor is pharmacologically characterized by the relative potency of a series of catecholamines and phenethylamines and by its susceptibility to specific blockade by phentolamine at relatively low concentrations (Ahlquist, 1966; Furchgott, 1967). Phentolamine, the most specific competitive alpha-adrenoceptor blocker currently available (Goodman and Gilman, 1975) and at a concentration considered to be specific for the alpha-adrenoceptor inhibited the serotonin induced contraction of the rat thoracic aortae, mesenteric artery and canine saphenous vein. From the experimental design, one can interpret that the serotonergic induced constriction is mediated through an alpha- adrenoceptor mechanism. In addition, phentolamine demonstrated similar pA£ values against norepinephrine and serotonin in the rat thoracic aortae and mesenteric arteries (Table 3). According to Arunlakshana and Schild (1959) and Furchgott (1957), this data must be interpreted to mean that the vasoconstrictor responses of these preparations to serotonin and norepinephrine are mediated by the same, or similar, alpha adrenergic receptors.

The interaction of serotonin and the alpha adrenergic receptor is probably a direct action on the vascular smooth muscle. The response of 174 the vasculature to serotonin was not diminished after presumed deple tion of norepinephrine by reserpine nor after amine reuptake inhibition by cocaine. Serotonin has been shown by Jarrott, et at, (1975) to be present in the sparsely innervated thoracic aorta (Burnstock, et at,

1972). The turnover of serotonin in the rat thoracic aorta and mesenteric artery was approximately twice the reported turnover for nor epinephrine (Spector, et at, 1972). This factor could explain the greater potentiation by cocaine of the contractile responses to sero tonin than to norepinephrine.

Reserpine pretreatment enhanced the receptor blocking activity of phentolamine against serotonin much more than against norepinephrine.

In the presence of cocaine, the pA2 values were less than that of control, possibly reflecting neuronal reuptake blockade. The blockade of reuptake by cocaine may result in a higher concentration of the agonists in the vicinity of the receptor. The agonists could then com pete more effectively with the antagonist resulting in the diminution of inhibitory activity of the antagonist. The converse would be true with reserpine.

Alternatively, and more logically, both reserpine and cocaine inter act with the calcium pools localized within the vascular smooth muscle cell membrane (Greenberg, et at, 1972; Bohr, et at, in press). The blocking activity of phentolamine is diminished by an increase in the extracellular calcium ion concentration. The effects of reserpine and cocaine could be secondary to their interaction with the calcium pool

involved with the antagonist-receptor interaction. 175

Thus serotonin, an indolealkylamine, appears to have a direct constrictor action on vascular smooth muscle by stimulation of an alpha adrenergic receptor. This stimulation is inhibited by phentolamine at concentrations considered to be effective for an alpha-adrenergic block ing agent. Serotonin satisfies the criteria set down for the definition of an alpha receptor stimulant (Ahlquist, 1966; Furchgott, 1967).

If the alpha-receptor is to be defined on the order of relative potency of a series of phenethylamines, which connotes a steric form of the receptor, then the definition would have to be broadened to include the more complicated serotonin molecule. Consequently, the steric specificity of the classical alpha-receptor should be able to conform to both phenethylamines and to the indolealkylamine. Kier (1960) suggests a three point attachment of serotonin, a steric configuration involving the two nitrogens and the hydroxyl group. This more complicated con figuration would be able to accommodate norepinephrine with its nitrogen and hydroxyl group. However, the order of potency does not fit in with the structural requirements for the primary configuration of the alpha receptor as one which would accommodate serotonin, but rather norep

inephrine. Since two different chemical series are now involved,

indolealkylamines as well as phenethylamines, an order of potency can no longer define the characteristics of an alpha receptor in the classical

sense. However, if the definition of an alpha receptor was based on the order of potency of an antagonist, such as phentolamine, a specific alpha blocker, then the nature of the alpha-receptor would encompass a broader spectrum. 176

Employing the definition of an alpha receptor stimulant as one which is susceptible to inhibition by phentolamine, the differentiation

of the blocking effect of tolazoline on the responses to norepinephrine

and serotonin suggests that the alpha receptor exists as two subtypes,

one with an affinity for norepinephrine (alpha-j) and one with an af

finity for serotonin {.alpha^) • The noradrenergic alpha receptor (a^)

site would be one possessing a high affinity and low capacity, whereas

the serotonergic alpha receptor (012) would have a low affinity but high

capacity binding sites for norepinephrine. The (*2 receptor would have high affinity for serotonin.

Tolazoline, purportedly a less specific a-adrenergic blocker, like

phentolamine is a 2 -substituted imidazoline and a partial adrenergic

agonist (Sanders, et al, 1975). Tolazoline produced a dose-related

shift to the right of the concentration effect curve of norepinephrine, whereas, it did not block the responses to serotonin

(Figure 12). Thus, tolazoline was able to differentiate between

the two subtypes of the alpha receptor, by occupying the high affinity,

low capacity binding site, thereby blocking the noradrenergic response.

The unoccupied, low affinity, high capacity (for norepinephinre) 0.2

sites were then more accessible to serotonin so that it was able to

elicit its contractile responses at the'lower concentrations. The tolazo

line induced depression of the maximum response to serotonin (Figure 12)

would suggest that few or no spare receptors of the 0 2 tyP® are present

for serotonin after adrenergic blockade with tolazoline. Alternatively,

since tolazoline is a partial agonist, some of the binding sites could 177 have been non-competitively or Irreversibly occupied, which would also cause a depression of the maximum response. Since serotonin is a weaker agonist, i.e., has a lower efficacy than norepinephrine, it should require a higher receptor occupancy to produce its response (Stephenson, 1956).

With few spare receptors available, the depression of maximum response

to serotonin would not be unexpected after adrenergic blockade with

tolazoline (Goldstein, et al, 1974). Alternatively, if the two subtypes are very similar in nature, i.e., both being blocked by phentolamine and

only susceptible to inhibition by tolazoline, there probably would be

some cross-reactivity of the two receptors. This could manifest itself

in a depression of the maximal response to serotonin.

Methysergide, a potent, relatively specific serotonergic blocker

(Goodman and Gilman, 1975), produced a dose related shift to the right

of the concentration effect curves for serotonin and norepinephrine.

This inhibition occurred in both rat thoracic aortae and the mesenteric

arteries. Thus, cross-reactivity occurs with two classical potent

antagonists, phentolamine and methysergide (Figures 9, 15), which

possess different chemical configurations. Both antagonists may possess

the ability to form similar steric configurations to adopt to the

receptors. This suggests that the receptor is less specific than the

a 2 receptor since tolazoline appears to.inhibit but not a2.

Phentolamine appears to have a high affinity as well as a high

intrinsic blocking activity for both alpha receptors, perhaps due to the 178

similarity of the subtypes, phentolamine blocks both and a£ receptors.

Alternatively, the steric configurational requirements for phentolamine to interact with the a receptors may not be rigid. Tolazoline appears to be able to differentiate the two alpha receptors. It was thought that by incubating the tissue preparations with tolazoline, we would be able to block the noradrenergic receptor (a^) and characterize, to a greater extent, the ot2 receptor. This crucial experiment could not be performed as rigorously as the previous experiments. Tolazoline plus cocaine caused the vascular strips to become unstable, with a resultant continuous potentiation of the responses to both agonists during the experiment (Figure 19). Imidizoline compounds inhibit monoamine oxidase

(Patil, 1976). The potentiation of the responses to the agonists may have been secondary to the decrease in the metabolism of the biogenic amines. Speculatively, prolonged blockade of ai receptors by tolazoline may result in de-repression of (*2 receptor protein synthesis, generation of more <*2 receptors and an enhanced response to serotonin and norepinephrine with time.

Prolonged exposure of the rat vasculature to tolazoline resulted in loss of methysergide-induced inhibition of the responses to serotonin and norepinephrine (Figure 18). If tolazoline inhibits the site,

this should allow methysergide to interact exclusively with the (X2 site,

then one would expect an even better blockade of the serotonergic 179

responses to methysergide. The converse in fact occurs. This cannot, at present, be explained until the effect of prolonged incubation with tolazoline on the stability of the vascular strips is explained.

Speculatively, methysergide may need both and (*2 sites open to mani fest its full blocking activity. Phentolamine however, possessed its blocking efficacy against serotonin. This data is difficult to interpret since tolazoline potentiated the vasculature strips during the course of the experiment. Nevertheless, the data strongly suggests that the receptor for serotonin is "alpha-receptor like" in nature.

If one assumes that the smooth muscle contraction induced by sero

tonin is related to the rate at which serotonin diffuses through the extracellular space and binds with the receptor but not the rate of entry into the cell (Handschumaker and Vane, 1967), the data from the binding study of 2-C^-serotonin adds preliminary support that phentola mine inhibits serotonin-induced contraction by inhibiting the binding of serotonin to its site of action. This data is in agreement with

other investigators (Thoa, et at, 1969; Born, 1962; Weiss and Rosecrans,

1971) and the results demonstrate a similarity to the contractility

studies performed with serotonin and phentolamine.

Tolzaoline neither inhibited the contractions induced by serotonin

nor the binding of 2-C^-serotonin. Phentolamine produced a dose 180 related Inhibition of the binding of 2-C^-serotonin (Figure 22), similar to that of the dose related inhibition of the contractile responses (Figure 9). Norepinephrine and cocaine inhibited the binding of serotonin. This probably occurred by direct competition for the receptor site by norepinephrine and by blocking the uptake neuronally and extraneuronally of serotonin by cocaine. This finding was similar to that of Thoa, et al> (1969) in which they inhibited the subcellular distribution of C^-serotonin in guinea pig vas deferens with norepi nephrine and cocaine.

Many theories have been put forward to explain the drug-receptor interaction. For a drug to elicit its response, a certain number of receptors have to be occupied (Stephenson, 1956). The events occurring beyond the drug-receptor interaction are still open to question. In

1964 Belleau proposed the macromolecular pertubation theory which sug gested that a conformational change in the proteins, which act as receptors, convert them from inactive species to ones capable of catalyzing reactions with substrates (Korolkovas, 1970). Speculatively,

tolazoline incubation of the vascular strips may cause some change in

the (*! receptor or protein to cause the receptor to catalyze some reac

tion and potentiate the responses with time. This biochemical reaction may be a by-product of the drug-receptor complex. This may be caused by a change in the free energy of the membrane, which is secondary to the

chemical modification of the receptor due to vibrational and electronic

forces. The alpha receptor may also exist as an allosteric enzyme which Monod and co-workers (1965) have proposed are polymers, made up of

one or more identical subunits and, therefore, can exist in at least two 181 different conformational states. Patil, et al, (1972) using the isomeric- activity ratios in rabbit, rat, cat and guinea pig aorta suggests a simi larity of the a-receptors. However, descrepencies existed in the activity with (-) norepinephrine. It is possible that the subtypes of the ot- receptor may show more rigid requirements in the (-) and (+) form for norepinephrine and that this may be a further avenue of research to characterize the and 0. 2 adrenergic receptors.

Serotonin ’and Canine Saphenous Vein

Cognizant of specie differences, the. canine saphenous vein was employed to gain further insight into the nature of the serotonergic receptor. As stated previously (see discussion on rat vasculature) , the criteria established and enumerated by Furchgott (1964, 1972) for the proper experimental conditions to analyze and identify adrenergic recep tors were stringently followed. Appropriate time controls for each ago nist and for each antagonist were employed. Equilibrium responses of the canine saphenous veins to serotonin and norepinephrine were obtained. The responses of the saphenous veins to the agonists were stable throughout the experimental procedures employed in the study. Experiments were per formed in the presence and absence of cocaine to inhibit the neuronal reuptake mechanisms involved in the potential termination of action of serotonin. Calcium disodium EDTA was always present in the physiologic

saline solition (PSS) to minimize oxidation of norepinephrine and

serotonin.

The presence of classical serotonergic receptors mediating

serotonin-induced venoconstriction was virtually ruled out when it was 182 apparent that methysergide, a potent serotonergic receptor antagonist, was without any inhibitory effect on the venoconstrictor response to serotonin. This finding is in agreement with the study of Clement and

Vanhoutte (1969) who speculated that the serotonergic receptor within the canine saphenous vein may not be a typical serotonergic receptor.

In agreement with their findings, the serotonergic receptor antagonist stimulated a contraction of venous smooth muscle. This contraction was inhibited by phentolamine. Clement and Vanhoutte (1969) also suggested that serotonin may elicit venoconstriction through alpha adrenergic rather than serotonergic receptors. These studies provide sufficient evidence to support this postulate, since classical serotonergic recep tors appear not to exist in the canine saphenous vein.

Cocaine, an inhibitor of the adrenergic neuronal reuptake of norepinephrine, enhanced the sensitivity of the canine saphenous vein to serotonin but depressed slightly the maximal response to this indole- alkyl amine. This finding of an enhanced sensitivity suggested that a mechanism involved in the termination of action of this venoconstrictor amine was impaired by cocaine. Serotonin does not release 7-^H- norepinephrine from the adrenergic nerves innervating the saphenous vein.

However, this finding does not rule out the possibility that the adren ergic nerve or some as yet undefined storage site, accumulates serotonin.

The experiment of Snipes, et at, (1968) employing rat vas deferens and electron-microscopy demonstrated the presence of an electron dense material in norepinephrine depleted adrenergic nerve terminals when the vas deferens were incubated with serotonin. Furthermore, cocaine decreases the accumulation of 2-C^serotonin in vasa deferentia (Thoa, 183 et al, 1969), mesenteric arteries and rat thoracic aorta (Curro, et al,

1976), perfused lung preparations (Junod, 1972) and blood platelets

(Gillis, et al, 1975). Thus cocaine-mediated enhancement of the veno constrictor responses to serotonin strongly suggest the presence of a reuptake mechanism in the saphenous vein for this indolealkyl amine and inhibition of the aforementioned mechanism by cocaine. It is doubtful that this enhancement is secondary to a cocaine-dependent non-specific sensitization of the smooth muscle cells. Previous studies demon strated that these low concentrations of cocaine did not sensitize the canine veins to potassium ion, a non-specific smooth muscle stimulant

(Bohr, et al, 1977; Somlyo and Somlyo, 1968).

Presumed inhibition of the inactivation mechanism for serotonin by cocaine resulted in a depression of the maximal contractile response to serotonin. Yet serotonin did not release norepinephrine. It is possible that serotonin may release a substance as yet undefined from the venous smooth muscle wall. Inhibition of the accumulation of serotonin elimi nates this component of the venoconstrictor response to serotonin.

Another explanation is that the presumably higher concentration of sero tonin at the receptor site may result in auto-inhibition of serotonergic mediated venoconstriction (Offermeier and Ariens, 1966). Auto-inhibition, a form of tachyphylaxis, has been described with high concentrations of serotonin in many smooth muscle preparations (Somlyo and Somlyo, 1970).

Speculatively, the higher concentrations of serotonin may interact with the tolazoline sensitive alpha adrenergic'receptor resulting in an apparent depression of the maximal response to serotonin (see below). 184

Many smooth muscle preparations, as well as the adrenal glands,

accumulate serotonin with an accompanying release of norepinephrine and

epinephrine respectively (Nishino, et al, 1970; Eble, et al, 1972).

Serotonin does not release 7-3H-norepinephrine from adrenergic nerves within the canine saphenous vein. The metabolism of 7-%-norepinephrine

is virtually unchanged from control values during superfusion with sero

tonin. Phentolamine inhibits the venoconstrictor responses to serotonin.

The inhibitory effect is still evident during presumed inhibition of

serotonin accumulation within the saphenous vein. Phentolamine mediated

inhibition of the venoconstrictor responses to serotonin is not secon

dary to deterioration of the vein during the experimental procedures.

The contractile response to serotonin are stable throughout the entire

experiment. In addition, an Arunlakshana-Schild plot of the antagonis

tic efficacy of phentolamine against serotonin and phentolamine against

norepinephrine generate parallel lines with different pA£ values. In

view of our adherence to the strict criteria advanced by Furchgott

(1964, 1972) for the differentiation of receptors (as discussed above),

the data presented support the conclusion that serotonin stimulates

alpha adrenergic receptors to elicit contraction of the canine

saphenous vein. Furthermore, these receptors appear to differ from

those of the classical adrenergic receptor.

Tto major criteria are established for the classification of

adrenergic receptors (see discussion on rat vasculature). The alpha adrenoceptor of smooth muscle may be pharmacologically characterized by the relative potencies of a series of related catecholamines and phenethylamine derivatives, necessary to elicit contraction of the 185 muscle preparations (Alquist, 1948), and the susceptibility of the responses to inhibition by low concentrations of phentolamine (Furchgott,

1972). By definition, serotonin is not a catecholamine nor a phenethylamine derivative, but rather an indole-alkylamine. Therefore, serotonin cannot be included in a series of this nature. This results in the necessity for characterization of the saphenous vein receptors for serotonin by evaluation of the effects of specific pharmacologic receptor antagonists on the venoconstrictor responses to serotonin.

Serotonin-induced contractions of canine saphenous veins are not inhibited by methysergide, a classic serotonergic receptor antagonist.

Venoconstrictor responses to serotonin were inhibited by phentolamine, the specific alpha adrenoceptor antagonist (Goodman and Gilman, 1975).

The pA£ of phentolamine against serotonin is less than that of phentol amine against norepinephrine. However, the slope of the logarithm of the (dose ratio-1 ) versus the logarithm of the phentolamine concentra tions are parallel. These findings are consistent with the conclusions that: (1 ) serotonin interacts with an alpha adrenoceptor to elicit its contractile response and (2 ) the alpha receptor differs from that of the noradrenergic receptor. This latter conclusion is supported by the findings with tolazoline, a purportedly less specific alpha adrenoceptor antagonist. Tolazoline is a histaminergic receptor stimulant as well as an alpha receptor antagonist. However, alpha adrenoceptor blockade by tolazoline selectively inhibits the venoconstrictor responses to norepinephrine yet potentiates the responses to serotonin. Following prolonged incubation of saphenous veins with tolazoline, the responses 186 to serotonin eventually stabilize. Under these conditions phentolamine is a more potent antagonist of serotonin than of the residual responses to norepinephrine. These findings add further support to the concept that the phentolamine sensitive receptor for serotonin differ from the classical receptors for norepinephrine, and may be a subclass of the classical alpha adrenergic receptor.

The mechanism of tolazoline-induced potentiation of the venoconstrictor responses to serotonin is unclear. Two possible explanations can be advanced but are speculative at the present time. Tolazoline is a weak inhibitor of type A monoamine oxidase, the enzyme involved in the deamination and eventual inactivation of serotonin and norepinephrine.

Inhibition of intra-and-extra-neuronal monoamine oxidase should inhibit

the inactivation of serotonin and norepinephrine and allow a greater concentration of these amines at their alpha receptors. This postulate

is questioned since serotonin, which theoretically should also compete with norepinephrine for monoamine oxidase and thereby diminish its

effectiveness, did not alter the metabolism of norepinephrine nor the

release of 7-3H-norepinephrine. This would suggest that deamination is not a major route of metabolism for serotonin in the canine saphenous

vein. More importantly, tolazoline-induced enhancement of the reponses

to serotonin occurred after presumed inhibition of neuronal reuptake and

potentiation of the venoconstrictor responses to serotonin with cocaine.

It is unlikely that tolazoline-induced inhibition of monoamine oxidase

can explain the potentiation of the venous smooth muscle responses to

serotonin. Alternatively, tolazoline-induced blockade of the 187 noradrenergic receptor may de-repress an Inhibitory regulatory mechanism on "alpha2 " receptors.

The data on skeletal muscle suggests that acetylcholine has an inhibitory effect on the development of extrajunctional receptors. It is possible, albeit highly speculative at the present time, that inhibition of "alpha^" receptors may increase the number of kinetic parameters of the alpha£ receptors. Alternatively, serotonin may act as a dilator substance when it interacts with tolazoline sensitive alpha receptors. Inhibition of these receptors may result in an ap parent tolazoline-mediated potentiation of the venoconstrictor responses to serotoi in, as a consequence of the inhibition of the depressor component of the response to serotonin.

Serotonin is synthesized and present in relatively high concentra tions in the walls of blood vessels and in platelets (Goodman and Gilman,

1975). This indolealkylamine is released into the circulation during vascular dammage and platelet aggregation and dysfunction (Harrison, et al, 1975) and possibly during stimulation of adrenergic nerves

(Myers, 1975). These observations and the results of the present study suggest that serotonin may play a role in the maintenance of venomotor and perhaps arterial smooth muscle tone via an alpha adreno ceptor mechanism at a time when sympathetic transmission is suppressed or inhibited. It is possible that during the hypotension of 188 anaphylaxis or during the early stage of hypertension associated with increased venous return, serotonin may act as a venoconstrictor through alpha adrenergic mechanisms to compensate for the reduced pressure or account for the venoconstriction, respectively, of these patho physiologic states.

In summary, serotonin-mediated contraction of canine saphenous veins are resistant to inhibition by methysergide. The venoconstric tor responses to serotonin are sensitive to inhibition by phentolamine, an alpha receptor antagonist. Another antagonist, tolazoline, failed to inhibit the venoconstriction responses to serotonin. The inhibitory effect of phentolamine was not secondary to serotonin- mediated release of norepinephrine since serotonin did not increase the efflux or decrease the metabolism of 7-^H-norepinephrine.

These findings clearly support the concept that serotonin-induced veno constriction is mediated through stimulation of alpha adrenoceptors on the venous smooth muscle cell membrane. The selective antagonism of norepinephrine by tolazoline in contrast to phentolamine clearly suggests the existence of two apparently similar, yet distinct alpha adreno ceptors in canine saphenous vein. Alternatively, serotonin and 189 norepinephrine may demonstrate different modes of binding to the same receptor.

The implication of serotonin in the origin of cerebral arterial spasm has led to possible further expansion of the above concept with

important clinical implications. The cerebral vessels of the cat are

supplied with sympathetic adrenergic nerves, originating in the superior

cervical ganglia. The cholinesterase-containing cholinergic nerves,

innervate only the pial vessles (Edvinsson and Owman, 1976). Serotonin

is a potent vasoconstrictor of isolated middle cerebral arteries from

cats and pial arteries from humans (Edvinsson and Owman, 1976). These vasoconstrictor responses are blocked by the serotonin antagonist, methysergide. Serotonin also induces a dose-dependent reduction in

cerebral blood flow in unanesthetized goats, which is partially

abolished by lysergic acid diethylamide (Lluch, et at, 1976). We have

found serotonin to be a vasoactive agent on the basilar and middle

cerebral arteries in the dog (Figure 41). The threshold to serotonin in

these vessels was about 10-fold higher than in the periphery. The

posterior cerebellar artery, branches of which supply the central noradrenergic neurons responded very poorly (data not shown). The basilar artery, branches of which supply the central serotonergic neurons,

did not respond as well as the middle cerebral artery. Auto-inhibition

is seen in the control response at 1X10“-*M serotonin. Phentolamine

caused a dose-dependent shift to the right of this auto-inhibitory

phenomena along with a shift to the right of the threshold response to

serotonin. At the higher doses a depression of maxima is observed. Thus 190

FIGURE

EFFECT OF ALPHA RECEPTOR BLOCKADE ON THE RESPONSE OF CANINE CEREBRAL VESSELS TO SEROTONIN (5-HT)

A typical record illustrating the inhibitory effect of phentolamine on serotonin-mediated vasoconstriction and auto-inhibition of constric tion in canine middle cerebral and basilar arteries. 191

effect OF/*LPHAR'^eerpJb ralLvessels to 5-HT

QN -TljE RESPONSE OF C ^ BASilar ARTERY CONTROL

1 m i n u t e

• m id d l e c e r e b r a l a r t e r y

5-HT K)

oomq PHENTOLAMINE 1*10 6m

B.A.

M.C.A.

PHENTOLAMINE I* I0 '7M

' &A. u. L ^ . 1

M.C.A.

PHENTOLAMINE 1*10 6M

aA .

PHENTOLAMINE 1*I0‘ *M

b a . 192 phentolamine is able to interact with the serotonergic-mediated vasoconstriction of canine cerebral vessels in a complex manner. The possible clinical implications of auto-inhibition of cerebral vessels to serotonin and its manipulation by the a-adrenergic blocking agent phentolamine could lead to a possible explanation of cerebral arterial spasm or migraine. TABLE 8

SUMMARY 194

TABLE 8. SUMMARY

Rat Vascular Smooth Muscle Studies

2. Serotonergic receptor blockade by methysergide inhibits the con tractile responses to both serotonin and norepinephrine.

3. These effects are not apparently due to release of vasoactive amines by either agonist since the effects occur despite amine depletion and blockade of the amine reuptake mechanism located within the adrenergic nerves.

4. Phentolamine, but not tolazoline, inhibits the accumulation of C^-serotonin by rat thoracic aortae. This effect is related to the concentration of phentolamine and parallels the inhibitory effect of phentolamine on serotonin-mediated contraction.

5. Phentolamine may interfere with receptor as well as non-receptor binding sites for serotonin.

6 . Serotonin-mediated contraction of rat thoracic aorta and mesenteric arteries may occur by stimulation of alpha receptors or by stimula tion of a receptor so similar to the alpha receptor that phentolamine cannot distinguish these receptors.

7. Alternatively, a component of the alpha receptor protein may be an integral moiety of the serotonergic receptor.

9. The instability of the rat vascular smooth muscle during prolonged incubation with high concentrations of tolazoline, a prerequisite for characterizing this putative receptor, make difficult further characterization of the receptors within these preparations. 195

TABLE 8. (Continued)

Canine Saphenous Vein Studies

2. A purportedly non-selective alpha receptor antagonist, tolazoline, selectively inhibits the contractile responses to norepinephrine but not serotonin.

3. During alpha receptor blockade by tolazoline, phentolamine is a more selective antagonist of the contractile responses to serotonin than to norepinephrine.

4. These effects are not secondary to serotonin mediated release of norepinephrine since serotonin does not significantly increase the release of 7-H^-norepinephrine or alter the metabolism of norepinephrine.

5. These findings clearly demonstrate that serotonergic mediated veno constriction is through stimulation of alpha-adrenoceptors on the venous smooth muscle cell.

6 . The selective antagonism of tolazoline, in contrast to that of phentolamine, clearly indicate the existence of two apparently different receptors sensitive to inhibition by phentolamine.

7. The data clearly indicate the existence of two subclasses of alpha adrenergic receptor in canine saphenous veins. 196

CONCLUSION

Evidence exists for the similarity of a-receptors (Patil, et al,

(1971) in rabbit aorta, vena cava, spleen and ileum, and rat vas

deferens and seminal vesicle. However, there has been considerably less

investigation of the pharmacological characteristics of responses

mediated b.y a-receptors in different tissues in comparison to responses

mediated by 3-receptors. Therefore, the conclusion that all a-receptors

are of a single type is highly tentative (Furchgott, 1972). The

evidence presented in this dissertation lends credence to the concept

that two subclasses of the a-receptor exist. This concept will have to

be tested in future experiments and by other investigators before a

perennial place in pharmacology literature is reserved.

/ 197

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