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Characterization of a [³H]-5- hydroxytryptamine binding site in rabbit brain

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Characterization of a [3H]-5-hydroxytryptamine binding site in rabbit brain

Xiong, Wen-cheng, M.S.

The University of Arizona, 1989

UMI 300 N.Zecb Rd. Ann Arbor, MI 48106

CHARACTERIZATION OF A [3H]-5-HYDR0XYTRYPTAMINE

BINDING SITE IN RABBIT BRAIN

Wen-cheng Xiong

A Thesis Submitted to the Faculty of the

COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE)

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE WITH A MAJOR IN TOXICOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 8 9 2

STATEMENT OF AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part made be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:

APPROVED BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

£21 dwne S David L. Nelson Date Associate Professor of Pharmacology and Toxicology To my husband, my parents, and brothers, with love. 4

ACKNOWLEDGMENTS

I wish to express my most sincere thanks to my advisor

Dr. David L. Nelson for his supervision, guidance and encouragement. Thanks goes to my graduate committee members Drs.

Hugh E. Lai/d II and Henry I. Yamamura for their advice and suggestions. Thanks are also given to Linda Cornfield, Anne

Killam, and Georgina Lambert for their assistance. Sincere appreciation goes to Anne Killam for her reading manuscripts and her time. 5

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS 7

LISTS OF TABLES 10

ABSTRACT 12

INTRODUCTION 13

History of 5-HT receptors 13 Pharmacological classification of 5-HT receptors 16 Second messengers of 5-HT receptors 23 Molecular biology of 5-HT receptors 27 Distribution of 5-HT receptors 31 Rationales of this study 31

METHODS 33

Preparation of membranes for ligand-binding assays 33 [3H]5-HT binding assay—saturation studies 33 [3H]5-HT binding assay—inhibition studies 34 [3H]5-HT binding assay—GTP and cation regulation 35 [3H]5-HT binding assay—NEM inactivation 35 [3HJ5-HT binding assay—Distribution of the 5-HT binding sites in rabbit brain 36 Data analysis 36 Materials 37

RESULTS 39

Blockade of 5-HT^ ant* 5-HTjq binding sites in rabbit CN 39 Saturation analysis of [3H]5-HT binding in rabbit CN 41 Comparison of the pharmacological profiles of [3H]5-HT binding sites in rabbit and bovine CN after blockade of 5-HTj^ and 5-HTjq sites 44 6

TABLE OF CONTENTS—Continued

Page

Effects of GTP on [3H]5-HT binding to rabbit and bovine CN 64 Interaction of GTP and cations on [3H]5-HT binding 70 Inactivation of the binding sites by NEM 80 Distribution of the [3H]5-HT binding sites in rabbit brain 81

DISCUSSION 86

CONCLUSIONS 94

REFERENCES 95 7

LIST OF ILLUSTRATIONS

Figure Page

1. 8-OH-DPAT and mesulergine competition studies with [3H]5-HT binding in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine in rabbit CN 40

2. [3H]5-HT saturation studies in rabbit CN in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine 42

3. Competition studies of 5-HT1A~selective

drugs with the non-5-HTiA/non-5-HTiQ binding sites in rabbit CN 46

4. Competition studies of 5-HTig-selective drugs with the non-5-HTjA/non-5-HTjQ binding sites in rabbit CN 47

5. Competition studies of 5-HTjQ-selective

drugs with the non-5-HTjA/non-5-HTjQ binding sites in rabbit CN 48

6. Competition studies of S-HTg-selective drugs

with the non-5-HTiA/non-5-HT;L0 binding sites in rabbit CN 49

7. Correlations between the pK| values of for 3 the non-5-HTjA/non-5-HTic [ H]5-HT binding sites in rabbit and the 5-HT^^, 5-HTjg, 5-HTjq, and 5-HT2 sites 50

8. Correlations between the pKj values of compounds for the non-5-HT1A/non-5-HT2c binding sites in rabbit and bovine CN 54

9. Inhibition curves for spiperone and spirilene against non-5-HT1A/non-5-HTlc [3H]5-HT binding in rabbit and bovine CN 56

10. Chemical structures of spiperone and its analogs 57 LIST OF ILLUSTRATIONS—Continued

Figure Page

11. Inhibition curves for ergonovine against 3 non-5-HT1A/non-5-HTlc [ H]5-HT binding in rabbit and bovine CN 60

12. Inhibtion curves for ergotamine and ergocryptine against the non-5-HTi^/non-5- HTjc [3H]5-HT binding in rabbit and bovine CN 61

13. Inhibtion curves for dihydroergotamine, dihydroergDcristine. and dihydroergocryptine against the non-5~HTiA/non-5-HTic [ 5- HT binding in rabbit and bovine CN 62

14. Effects of GTP on the non-5-HT^A/non-5-HTjQ [3H]5-HT binding in rabbit and bovine CN without calcium 65

15. Effect of GTP on agonist inhibition of [3H]5-HT binding to non-5-HTiA/non-5~HTic sites in rabbit and bovine CN without calcium 66

16. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in rabbit CN without calcium 67

17. [3H]5-HT sturation studies in the presence and absence of 100 uM GTP in bovine CN without calcium 68

18. Effects of GTP on the binding of [3H]5-HT in the precence of 100 nM 8-OH-DPAT and 100 nM mesulergine with or without calcium 72

19. Effects of GTP on agonist inhibition of [3H]5-HT binding to non-5-HTiA/non-5~HTic [3H]5-HT binding in rabbit and bovine CN in the presence of 3 mM calcium 74 9

LIST OF ILLUSTRATIONS—Continued

Figure Page

20. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in the rabbit with 3 mM calcium 75

21. [3H]5-HT saturation studies in the presence and absence of 100 uM GTP in the bovine CN with 3 mM calcium 76

22. Cation effects on the non-5-HT1A/non-5-HTjQ [3H]5-HT binding sites in rabbit and bovine CM membranes 78

23. NEM sensitivity of the non-5-HTj^/non-5-HTjQ [3H]5-HT binding site in bovine and rabbit CN membranes 83

24. Regional distribution of specific [3H]5-HT binding in the absence or presence of 100 nM 8-OH-DPAT and 100 nM mesulergine in rabbit brain membranes 84 10

LIST OF TABLES

Table Page

1. Drug affinities for 5-HT1A, 5-HT1B, 5-HTlc, 5-HT^q, and 5-HT2 receptors 17

2. Results of saturation experiments performed in rabbit and bovine CN with [3H]5-HT in the absence and presence of 100 nM 8-OH-DPAT and 100 nM mesulergine 43

3. Correlation between the non-5-HTjA/non-5- HTic [3H]5-HT binding in the rabbit CN and 5-HT1A, 5-HTjg, 5-HT^q, and 5-HT2 binding 51

4. Affinity values of drugs for [3H]5-HT binding sites in rabbit CN in comparison to bovine brane 5-HT1D sites 53

5. Computer-assisted nonlinear regression analysis of spiperone , spirilene and spiramide interactions with [3H]5-HT binding in rabbit and bovine CN 58

6. Computer-assited nonlinear regression analysis of some ergots and dihydroergots

interaction with non-5-HTjA/non-5-HT2Q binding in rabbit and bovine CN 63

7. Results of saturation experiments performed in the rabbit and bovine CN to the non-5- HTiA/n°n-5-HTic [3H]5-HT binding in the presence and absence of 100 uM GTP 69

8. Results of saturation experiments performed in the rabbit and bovine CN to the non-5- 3 HT1A/non-5-HTlc [ H]5-HT binding in the presence and absence of 100 uM with 3 mM calcium 77

9. Effects of cations on regulation of the 3 non-5-HT1A/non-5-HTic [ H]5-HT binding by GTP 79 11

LIST OF TABLES—Continued

Table Page

10. Regional distribution of specific [3H]5-HT binding in the absence or presence of 100 nM 8-OH-DPAT and 100 nM mesulergine 85 12

ABSTRACT

In the present study non-5-HT1A/non-5-HT1Q binding sites in the rabbit caudate nucleus (CN) were examined to determine if they might be identical to the recently discoverd 5-HT1D sites in the bovine CN. The characterizations were carried out measuring high-affinity [3H]5-HT binding under conditions where 5-HTja and

5-HTjq sites were pharmacologically masked in both tissues.

Comparison of the pharmacologic profiles of the bovine 5-HTjq and rabbit non-5-HT1A/non5-HTlc sites revealed similarities, but showed distinct differences. For example, spiperone gave a Kj value of 2.8 uM in the rabbit CN, while concentrations of up to

100 uM did not produce even 50% inhibition of [3H]5-HT binding in the bovine CN. [3H]5-HT binding in the bovine CN was significant ly more sensitive to inhibition by GTP than was [3H]5-HT binding in the rabbit CN, and this effect was differentially sensitive to calcium and other divalent cations (i.e., Mg2+, Mn^+) in the two tissues. [3H]5-HT binding in the bovine CN was significantly more sensitive to inhibition by NEM than it was in the rabbit CN.

3 Thus, It may be concluded that the non-5-HTjA/non5-HTjC [ H]5-HT binding sites in rabbit CN are distinct from those in the bovine

CN, and we propose that they be tentatively identified as 5-HTj^ to distinguish them from the 5-HTjp site. 13

INTRODUCTION

History of 5-HT receptors

5-hydroxytryptamine (5-HT, serotonin) was isolated and

chemically characterized nearly four decades ago, and is now

generally accepted to function as a neurotransmitter and neuromod-

ulatory agent. Early research focused on the measurement of

content, synthesis, and metabolism of 5-HT, and only recently has

the focus shifted to characterization of 5-HT receptors. In 1957,

Gaddum and Picarelli showed that at least two distinct 5-HT

effects were observed in the smooth muscle of guinea-pig ileum.

The first type of 5-HT activity could be blocked by dibenzyline

(i.e., phenoxybenzamine) and was designated the "D" receptor. A

second type of 5-HT receptor involved an indirect contractile

effect of 5-HT, which occurred via neuronal activation of

acetylcholine release and could be inhibited by morphine. These

sites were designated "M" receptors. At that time, it was felt

that M receptors were found primarily in the peripheral nervous

tissue and D receptors In the smooth muscle (Gaddum and Picarelli,

1957). Later studies demonstrated that the effects of morphine

and dibenzyline are not directly mediated by 5-HT receptors, so

that these drugs are of minimal use in the current classification

system of 5-HT receptors. The possibility of dual 5-HT receptors

was not explored systematically or successfully until 14

the past decade (Humphery, 1984). However, these classic studies first demonstrated the existence of at least two distinct 5-HT receptors.

The development of radioreceptor binding assays during the past ten years represents a major breakthrough in pharmacolog ical methodology (Snyder, 1978). Early pharmacological studies relied on biological assays to measure drug potencies. Inherent problems with such assays included the determination of drug concentration at receptor sites, specificity of drug action, and correlation with other biological systems. By contrast, the use of radioligand studies has eliminated most of these problems.

The first attempt to label 5-HT receptors in the central nervous system occured before the development of rapid filtration techniques (Marchbanks, 1966, 1967; Fiszer and De Robertis, 1969).

Farrow and Van Vanukis (1972) attempted to label 5-HT receptors with [3H]LSD. A second major development in the study of central

5-HT receptors was the use of [3H]5-HT in radioreceptor assays

(Bennett and Snyder, 1976). However, although the pharmacological profiles of both [3H]5-HT and [3H]LSD binding suggested the label ing of 5-HT receptors, important differences were noted. For instance, d-LSD was one of the few drugs tested that had similar affinity for both [3H]5-HT and [3H]LSD sites. The agonist 5-HT, on the other hand, had almost 100-fold greater affinity for

[3H]5-HT sites than [3H]LSD binding sites. At that time, Bennett 15

and Snyder (1976) suggested that [3H]5-HT and [3H]LSD bound to two different "states" of the 5-HT receptor in brain membranes. A third [3H]ligand available to study 5-HT receptor was [3H]spiperone. However, marked differences were noted between the binding characteristics of [3H]5-HT, [3H]LSD, and [3H]spiperone. In 1979,

Peroutka and Snyder concluded that at least two distinct 5-HT membrane recognition sites exist in the central nervous system.

The sites labeled by [3H]5-HT were designated "5-HT} receptors", and those labeled by [3H]spiperone were designated "5-HT2 recep tors Since [3H]LSD had equal affinity for both sites, it was proposed that this ligand could be used to label both 5-HTj and

5-HT2 receptors. 5-HTj binding sites labeled by [3H]5-HT were soon shown to be heterogeneous. Nonsigmoidal inhibition of

[3H]5-HT by spiperone led to the suggestion that sites with high affinity for spiperone should be designated 5-HTjA, while sites with relatively low affinity for spiperone should be designated

5-HTsites (Nelson et al., 1981). The 5-HTlc binding site was later discovered as a consequence of autoradiographic studies using [3H]5-HT (Pazos et al., 1984). Most recently, the 5-HTjd binding site has been identified in bovine, pig, and human brain membranes (Heuring and Peroutka, 1987; Hoyer et al., 1988).

Although the "D" receptor of Gaddum and Picarelli (1957) appears to coincide with the 5-HT2 binding site, the "M" receptor has not yet been linked with a radioligand binding site in brain 16

membranes. Therefore, it has been proposed that the receptors mediating the effects of 5-HT at putative "M" receptors be desig nated "5-HT3" receptors (Bradley et al., 1986). Later, the 5-HT 3 receptors were found in the rat brain by Kilpatrick et al. (1987).

Most recently, a novel 5-HT receptor has been proposed by Dumuls et al. (1988). The pharmacological characteristics of this recep tor do not correlate with previously defined subtypes of 5-HT receptors. Therefore, this receptor has been designated the "5-

HT4" receptor.

Pharmacological characterization of 5-HT receptors

Since the initial classification of central receptors for serotonin into two principal subtypes, i.e., 5-HTj and 5-HT2

(Peroutka and Snyder, 1979), there has been a dramatic increase in the number of proposed additional receptor types for this neuro transmitter. This is especially the case for the 5-HTj subtypes, which have been defined on the basis of their high affinity for

5-HT itself. Currently, the proposed subtypes in this class include 5-HT1A (Pedigo et al., 1981; Gozlan et al. , 1983), 5-HTjq

(Pedigo et al., 1981; Hoyer et al., 1985a), 5-HT^e (Pazos et al.,

1984; Yagaloff and Hartig, 1985), and 5-HTjq (Heurlng and Perout ka, 1987) in the central nervous system, as well as the 5-HTjp subtype (Mawe et al., 1986) in the periphery. The pharmacological properties of these subtypes are as follows (Table 1). 17

Table 1. Drug affinities for 5-HTj^, 5-HT1B, 5-HTlc, 5-HT1D, and 5-HT2

receptors

Kj(nM) 5-HT1A 5-HT1B 5-HTlc 5-HT1D 5-HT2

<10 5-CT RU 24969 Mesulergine 5-CT Spiperone

8-OH-DPAT 5-CT Metergoline 5-HT Mesulergine

5-HT 5-HT Methysergide Metergoline Mianserin

RU 24969 Metergoline

d-LSD Methysergide

10-1000 Metergoline Metergoline Mianserin Methysergide d-LSD

Methysergide Methysergide 5-HT mianserin

Spiperone d-LSD RU 24969 8-OH-DPAT

Mesulergine 5-CT d-LSD

d-LSD RU 24969

>1000 Mianserin Mianserin Spiperone Mesulergine RU 24969

Spiperone 8-OH-DPAT Spiperone 5-HT

Mesulergine 8-OH-DPAT

8-OH-DPAT

Abbreviations used: 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 8-

hydroxy-2-(di-n-propylamino)tetralin; RU 24969, 5-methoxy-3-(l,2,3,6-

tetrahydropyridin-4-yl)-14-indole succinate; d-LSD, d-lysergic acid

diethylamide. 18

3-HTjA receptors

A number of radioligands have been shown to label the

3-HTjA receptor. Although the 5-HTjA site can be labeled with

[3H]5-HT, it can be more directly labeled with

[3H]8-hydroxy-2(di-n-propyl-amino)tetralin (8-OH-DPAT) (Gozlan et al., 1983, Hall et al., 1985, Peroutka, 1986, Hoyer et al.,

1985b), [3H]ipsapirone (formerly called TVX Q 7821) (Dompert et

;il . , 1985), [3H]buspirone (Moon & Taylor 1985) and [3H]l-(2-(4- aminophenyl)ethyl)-4-(3-trifluro-methylpheny)piperazine (PAPP)

(Asarch et al., 1985). In addition [3H]WB 4101, previously con sidered a selective alphaj-adrenergica radioligand, has been demonstrated to label the 5-HTjA site (Norman et al., 1985).

Regardless of the [3H]ligand used to label the site, it displays high and selective affinity for 8-OH-DPAT, 5-carboxamidotryptamine (5-CT), 5-methoxydimethyltryptamine, ipsapirone, and buspirone. In addition [3H]8-0H-DPAT has been reported to label the "5-HT transporter" (Schoemaker and Langer, 1986).

5-HTjg receptors

The putative 5-HTjg site has been more difficult to be characterized. Sills et al. (1984) defined 5-HTjg binding site as specific [3H]5-HT binding in the presence of 1 mM GTP and 2000 nM spiperone. They concluded that RU 24969 and TFMPP were selective

5-HT1B agents. However, these initial selectivity studies have 19

not been confirmed until the 5-HTjb site was more directly la beled in rat brain with [125I]cyanopindolol (Pazos et al 1985,

Hoyer et al., 1985a). The [125I]cyanopindolol site has high affinity for 5-HT and RU 24969 and relatively low affinity for d-

LSD and 8-OH-DPAT.

The 5-HT^g site has been associated with 5-HT, acetylcho line, and glutamate "autoreceptors" in rat brain. Depolarization of the neurons by potassium or electrical stimulation causes release of the pre-stored neurotransmitter. The release can be inhibited by 5-HT and related agonists, presumably though a

"presynaptic autoreceptor." Engel and colleagues (1986) demonstrated that no significant correlation was observed between drug potencies at the 5-HT "autoreceptor" and drug affinities for

5-HTlc or 5-HT2 binding sites. Significant correlations were ob tained with drug affinities for both 5-HT1A and 5-HT1B sites.

However, selective 5-HT1A drugs such as 8-OH-DPAT and ipsapirone had no effect on the autoreceptor and therefore were not used in the calculation of the correlation coefficient. As a result, the receptor mediating release of 5-HT from nerve terminals is mediat ed by the 5-HTjg binding site in rat brain. A similar conclusion was reached from an analysis of 5-HT and related drug effects on the release of [^Hjacetylcholine from rat hippocampal nerve end ings (Maura & Raiteri, 1986) and on the release of endogenous glutamate induced by depolarization of rat cerebellum synaptosomes 20

(Raiteri et al., 1986).

5-HTic receptors

The 5-HTjc site was first characterized in membranes from pig choroid plexus and cortex (Pazos et al., 1984; Hoyer et al. , 1985b). The site was labeled by both [3H]5-HT and [3H]mesu-

lergine. Independently, Yagaloff & Hartig (1985) labeled the site with [125I]LSD in the rat choroid plexus. The site has high affinity for 5-HT, methysergide, and mianserin and relatively low affinity for RU 24969. These pharmacological characteristics are also shared with the putative 5-HTjq site identified in rat cortex

(Hoyer et al., 1985b; Peroutka, 1986).

5-HTjd receptors

Recently, a fourth subtype of 5-HTj site labeled by

[3H]5-HT has been identified in bovine brain membranes (Heuring &

Peroutka, 1987). The addition of either the 5-HT1A-selective drug

8-OH-DPAT (100 nM) or the 5-HTlc-selective drug mesulergine (100 nM) to the radioligand assay results in a 5-10% decrease in spe cific [3H]5-HT binding. Competition studies using a series of

pharmacological agents reveal that the sites labeled by [3H]5-HT in bovine caudate in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine appear to be homogeneous. 5-HT1A~selective agents such as 8-OH-DPAT, ipsapirone, and buspirone display micromolar 21

affinities for these sites. 5-HTig-selective drugs RU 24969 and

(-)pindolol are approximately two orders of magnitude less potent

at these sites than at 5-HTjg sites that have been identified in

rat brain. Agents that display nanomolar potencies for 5-HTlc

sites such as mianserin and mesulergine are two to three orders of

magnitude less potent at these binding sites in bovine caudate.

In addition, both 5-HTo and 5-HT3-selective agents are essentially

inactive at these binding sites. These [3H]5-HT sites display

nanomolar affinity for 5-CT, 5-methyoxytryptamine, metergoline,

and 5-HT.

5-HT2 receptors

In contrast to the subtypes of the 5-HTj sites, 5-HT2

sites appear to be homogeneous. [3H]spiperone, [3H]LSD,

[3H]mianserin, [3H]ketanserin, [3H]mesulergine, [3H]n-

methylspiperone can be used to label the 5-HT2 binding site

(Leysen, 1981; Leysen et al., 1978 and 1982; Peroutka & Snyder,

1983; Pazos et al., 1984 ). Serotonergic antagonists have high

affinity for this site while 5-HT and related tryptamines are

markedly less potent. Recently, the hallucinogen [3H]-D0B has

been synthesized and reported to label a high affinity "state" of

5-HT2 receptor in the central nervous system (Titeler et al. ,

1985; Titeler & Lyon, 1986; Lyon et al., 1987). Later, Peroutka

et al. (1988) used (R)-(-)-[77Br]DOB to study these sites, and 22 they found that [77Br]DOB site did not appears to be a high affinity "state" of the 5-HT2 receptor but may label a subset of heterogeneous 5-HT2 recognition sites. Studies are in progress to determine the relevance of this site to 5-HT2 sites labeled by

[3H]antagonists.

5-HT3 receptors

The pharmacological characteristics of the 5-HT3 recep

tors are quite distinct from the 5-HTj and 5-HT2 binding sites identified in the brain. Receptors of the 5-HT3 type have been characterized on isolated peripheral tissue models such as the rat vagus nerve, guinea-pig ileum and isolated rabbit heart. In general, 5-HT effects in the 5-HT3 receptors are not affected by

5-HTj and/or 5-HT2-selective drugs such as 8-OH-DPAT and ketanserin. Nevertheless, 5-HT has multiple effects in the 5-HT3 recep tors that can often be blocked with drugs such as MDL 72222, (-

)cocaine, metoclopramide, GR 38032F, and ICS 205-930 (Fozard,

1984; Richardson et al., 1985; Richardson and Engel, 1986).

Recently, Kilpatrick et al. (1987) reported direct evidence for

the existence of 5-HT3 receptors in rat brain tissue, based on high affinity binding of the potent 5-HT3 receptor antagonist

[%]GR 65630 to homogenates of rat entorhinal cortex.

5-HT4 receptors 23

Recently, 5-HT4 receptors have been found in mouse embryo colliculi neurons in primary culture (Dumuis et al. , 1988, Dumuis et al., 1989). The pharmacological profile characterized with agonists and antagonists suggests that this 5-HT receptor does not appear to correspond to a known 5-HT receptor. On this 5-HT receptor, 5-HT (ECgo= 109 ± 17 nM), 5-MeOT and BRL 24924 (Dumuis et al., 1989) are equipotent agonists to stimulate adenylate cyclase. The selective drugs for the 5-HTj^, 5-HT1B, 5-HTlc, 5-

HTjd. 5-HT2, and 5-HTg receptors are almost inactive in reversing

the 5-HT stimulating effect. The selective 5-HT3 antangonist ICS

205 930 is a full competitive antagonist at this receptor with Kj about 1000 nM. A receptor with similar characteristics has also

been found in guinea pig hippocampal membranes. In these mem

branes, the second receptor of low affinity for 5-HT, termed Rl, which is positively coupled to adenylate cyclase, is also antago nized by ICS 205 930.

Second messengers of 5-HT receptors

The activation of 5-HT receptors leads to stimulation or inhibition of adenylate cyclase activity (Enjalbert et al., 1978;

Peroutka et al., 1981), activation of phosphoinositide turnover

(Pazos et al. , 1984; Hoyer et al., 1985b), and direct activation of ion channels (Yankel, J.L. and Jackson, M.B., 1988). Some 5-

HT receptors, such as 5-HTj , 5-HT2 and 5-HT4 receptors are of the 24

G protein-associated super-gene family, which includes rhodopsin, adrenergic receptors, muscarinic receptors, dopamine receptors, substance K receptors, and angiotensin receptors. G proteins are a group of membrane-associated guanyl nucleotide binding proteins which are heterotrimers, with subunits designatedoC,/8 and f . G proteins function as transducers in the relay of the signal to an effector molecular. The functional diversity in the family of G proteins resides in the oc subunit of the oligomeric complex(<*^0 . which regulates the activity of a number of effector molecules, such as adenylate cyclase (Stryer and Bourne, 1986; Gilman, 1987), phopholipase C (Cockcroft and Gomperts, 1985), cyclic GMP-phosphodiesterase (Stryer et al., 1981), and ion channels (Pfaffinger et al. , 1985; Holz et al., 1986). The binding of GTP to specific sites on G proteins both activates effectors and decreases the affinity of the receptors for their respective agonists. Evi dence for the G protein involvement in the 5-HT receptor-second messenger coupling includes: decrease of 5-HT agonist (but not antagonist) binding affinity by guanyl nucleotides (Hall et al.,

1985; Schlegel and Peroutka, 1986); imitation of 5-HT responses by agents which could directly stimulate G proteins, including

Gpp(NH)p and GTP^JS, and sensitivity of 5-HT responses to specific bacterial toxins such as pertussis toxin or cholera toxin (Andrade et al., 1986; Zgombick et al., 1989). The sensitivity of [3H]5-HT binding to GTP is significantly decreased in the presence of 25 calcium both in 5-HTj binding sites measured in rat brain (Mallat and Hamon, 1982), and 5—HTjjj binding sites measured in bovine caudate nucleus (Heuring and Peroutka, 1987).

5-HT receptors which are coupled to adenylate cyclase include 5-HTjA, 5-HTjg, 5-HTjj), and 5-HT4 receptors. Radioligand binding data support an association between 5-HT1A receptors and a

5-HT-sensitive adenylate cyclase. Regulation of neurotransmitter binding by guanine nucleotides often reflects an association of the agonist binding site with an adenylate cyclase. Thus, GTP and

GDP inhibit the binding of [3H]8-OH-DPAT to brain homogenates

(Hall et al., 1985; Schlegel & Peroutka, 1986). In addition, guanine nucleotides significantly reduce agonist potencies for

[3H]8-0H-DPAT binding sites whereas antagonist potencies are not affected by nucleotides. In studies using both rat (Markstein et al., 1986) and guinea pig (Shenker et al., 1985) hippocampal membranes, a 5-HT-sensitive adenylate cyclase could be stimulated by nanomolar concentrations of 5-HT1A selective agents such as 5-

CT and 8-OH-DPAT. In addition, 5-HT can inhibit forskolin-stimulated adenylate cyclase in rat and guinea pig hippocampal mem branes (De Vivo & Maayani, 1986). The 5-HT1A receptor also appears to mediate inhibition of VIP-stimulate cyclic AMP formation in purified striatal and cortical cultured neurons (Weiss et al. ,

1986). Thus, the 5-HT1A receptor appears to modulate adenylate cyclase activity in certain brain regions. Recently, it has been 26

found that 5-HTjA not only modulate adenylate cyclase, but also

inhibit carbachol-stimulated phosphoinositide turnover (Nicoletti

et al., 1986; Claustre et al. , 1988), and activate some ion

channels, such as K+ channels (Andrade et al., 1986). Recently,

5-HT1B and 5-HTjq receptors have been found to negatively couple

to the adenylate cyclase (Bouhelat et al., submitted, Hoyer and

Schoeffter, 1988). 5-HT4 receptors positively couple to the

adenylate cyclase (Dumuis et al., 1988).

Besides adenylate cyclase, phosphoinositide turnover is

believed to be a common "second messenger" in the transduction of neurotransmitter signals to the cell effector. The hydrolysis of

phosphoinositides may modulate a number of intracellular processes

including calcium flux, increased arachidonate metabolism, in

creased cyclic GMP production , and protein kinase C activation.

5-HT receptors which have been shown to increase phosphoinositide

turnover include 5-HTjq and 5-HT2 receptors. There is good evi

dence that 5-HTjq receptor-activation in rat choroid plexus leads

to the stimulation of phospholipase C and to the breakdown of

inositol phospholipids (Conn and Sanders-Bush, 1986; Conn et al.,

1986). The effects of a series of agonists and antagonists on

inositol 1-phosphate (IPj) accumulation in pig choroid plexus

homogenates have been studied (Pazos et al., 1984; Hoyer et al.,

1985b). There was a highly significant correlation between the

rank order of potency of agonists and antagonists and their affin 27 ities to 5-HTjq sites. Moreover, potent agonists at 5-HTj^, 5-

HT-^g and 5-HTjq receptors were only effective at high concentra tions. 5-HT2 and 5-HT3 receptor antagonists (i.e., ketanserin and

ICS 205-930) failed to inhibit the effects of 5-HT at low concen trations, confirming an effect mediated though a 5-HTjc receptor.

5-HT-induced phophoinositide hydrolysis in rat cerebral cortex

(Kendall & Nahorski, 1985; Conn & Sanders-Bush, 1985) appears to occur as a result of 5-HT2 receptor activation. 5-HT stimulates phosphoinositide turnover with an EC50 of 1 uM. The response to

5-HT is blocked by nanomolar concentrations of ketanserin, and phosphoinositide turnover is not affected by 8-0H-DPAT. Further more, tricyclic antidepressants decrease both 5-HT2 binding sites and 5-HT-induced phosphoinositide turnover (Conn & Sanders-Bush,

1986).

In addition, some 5-HT receptors, such as the 5-HT3 receptors, are of ion channel super-gene family, which includes

nictinic acetylcholine receptors, GABAA receptors, and glycine receptors. 5-HT binds to the 5-HT3 receptor to activate the ion channels directly to mediate the physiological responses (Yankel,

J.L. and Jackson, M.B., 1988).

Molecular biology of 5-HT receptors

Molecular biological techniques have made it possible to study the 5-HT receptors at the molecular level. Three 5-HT 28

receptor subtypes (5-HTlc, 5-HT1A, and 5-HT2) have been cloned by

three different laboratories using three different approaches.

All three receptors are single subunit proteins and members of the

G-protein receptor superfamily (Dohlman et al., 1987). This

receptor family is characterized by the presence of seven trans

membrane domains in each receptor and the ability to activate G-

protein-dependent processes, induing activation or inhibition of adeneylate cyclase activity, activation of phosphoinositide turn

over and direct activation of ion channels.

The first 5-HT receptor to be cloned was the 5-HTjq receptor. Injection of 5-HTjq receptor mRNA into Xenopus Oocytes

leads to the appearance of a large Ca2+-activated Cl~ conductivity

increase in response to the application of 5-HT (Lubbert et al.,

1987a). This signal was used in a hybrid depletion screening

strategy which was successful in isolating a 1.9 Kb fragment of

mouse tumor 5-HTlc receptor cDNA (Lubbert et al., 1987b). Subse

quently, another group used the oocytes to screen RNA transcribed

in vitro from a rat choroid plexus cDNA library to isolate a 3 Kb

cDNA clone containing the entire coding region of the 5-HTjc receptor (Julius et al., 1988).

The next 5-HT receptor to be cloned, the 5-HTi^ receptor, was obtained from an unexpected direction, and by a very different strategy. In 1987, an intronless gene was isolated by screening a

human genomic library at low stringency with a full-length beta2- 29 adrenoceptor clone (Kobilka et al., 1987). At that time, the function of the clone (designated G-21) was not known. Subse quently, it was discovered that transfection of the G-21 clone into monkey kidney cells produces a protein that exhibits the ligand-binding characteristics of the 5-HT1A receptor (Fargin et al., 1988). Transfection of this clone into a functional response system is eagerly awaited, since some groups have observed inhibi tion of adenylate cyclase activity by the 5-HTjA receptor (Weiss et al., 1986; De Vivo and Maayani, 1986), while other groups have observed activation of adenylate cyclase activity (Markstein et al., 1986; Shenker et al., 1985). Transfection and reconstitution studies should soon help clarify the the possible second messenger couplings of this receptor. The fact that the 5-HTja receptor was obtained by hybridization to a beta2-adrenoceptor clone is re flected in the strong nucleotide sequence homologies that exist between these two receptors.

The latest addition to the family of 5-HT receptor clones is the 5-HTg receptor. This receptor was obtained by screening a rat forebrain cDNA library with oligonucleotides derived from sequence data on the 5-HTiq on the 5-HTjq receptor (Pritchett et al., 1988). Similarities in ligand binding properties and second messenger coupling of the 5-HT2 and 5-HT^q receptors have long been apparent (Hoyer, 1988). This encouraged a cloning strategy based on presumed amino acid sequence homologies between the two 30

receptors. The cloned 5-HT2 receptor was transfected into human embryonic kidney cells, where it exhibited ligand binding proper ties in general agreement with binding data from rat cortical membrane studies. The affinity of the transfected receptor for spiperone, ketanserin and mianserin ranged from 0.5 nM to 1.8 nM, in good agreement with previous studies. The transfected 5-HT3 receptor was able to raise intracellular Ca^+ levels upon addition of 5-HT, as expected from previous data on the coupling of the 5-

HT2 receptor to the phosphoinositide second messenger system

(Sanders-Bush and Conn, 1986). In addition, RNA produced by in vitro transcription of 5-HT2 receptor cDNA induced a CI- conduc tivity increase in response to 5-HT three days after injection into Xenopus oocytes.

More recently, An approach based on the polymerase chain reaction has been devised to clone new subtypes of 5-HT receptors.

Libert et al. (1989) degenerated primers corresponding to consensus sequences of the third and sixth transmembrane segments of available receptors to selectively amplify and clone the receptors of G protein supergene family from thyroid cDNA library.

They obtained the clones of 5-HT1A and a new receptor, which has close structural similarity to 5-HT1A receptor. Studies are in

progress to determine whether or not this gene encode a member of the large family of 5-HT receptors. 31

Distribution of 5-HT receptors

The different subtypes of 5-HT receptors are distributed

unevenly in the brain. The 5-HTj^ site is densely present in the

CA1 region and dentate gyrus of the hippocampus and in the raphe nuclei (Pazos and Palacios, 1985; Hoyer et al., 1986a). The

highest densities of 5-HT1B sites in rat brain are found in the globus pallidus, dorsal subiculum and substantia nigra (Pazos and

Palacios, 1985). Recent data has demonstrated that this site can

also be labeled with [3H]5-KT in rat frontal cortex (Peroutka,

1986; Blurton and Wood, 1986). Moreover, the 5-HTjg site appears

to be species-specific in that it is present in rat and mouse

brain but not in guinea pig, cow, chicken, turtle, frog, or human

brain membrHnes (Henri rig c:t. al . , 3986). The 6-HTjq has only been characterized in membranes from choroid plexus and cortex (Pazos

et al. . 1984a and b; Hoyer et al. , 1985b). Regional studies of

5-HTjp demonstrated that this class of sites is most dense in the

basal ganglia but exists in all regions of bovine, rat, pig, and human brain (Heuring and Peroutka, 1987; Hoyer et al., 1988).

The highest level of 5-HT2 binding is in layer IV of the cerebral cortex and caudate, with all other brain regions having substantially fewer binding sites (Pazos et al., 1985; Hoyer et al., 1986b).

Rationales of this study 32

The ability of the drug spiperone to discriminate between subtypes of 5-HTj sites defined by [%]5-HT binding was studies previously in our laboratory (Schnellmann et al., 1984). In certain species, i.e., the cat, rabbit, and guinea pig, there appeared to be a [3H]5-HT binding site that corresponded to nei ther the 5-HTjA nor 5-HTjg classifications that had been defined in the rat. More recently, investigators have demonstrated that the 5-HT1B site is, in fact, present in only a restricted number of species, i.e., the rat and mouse (Hoyer et al., 1985; Heuring et al. , 1986) and that in certain other species a similar, but distinct, site designated 5-HTio exists (Heuring and Peroutka,

1987). This suggested the possibility that the non-5-HTiA/non-

5-HTib site seen in our previous study was actually the 5-HTjq site. However, a discrepancy existed in that the Kj of spiperone for the non-5-HTi^/non-5-HTi3 site in our previous study was in the range of 0.4-2.4 uM, while the reported for spiperone at

5-HTjd site in the bovine caudate nucleus (CN) was greater than

100 uM, Thus, the present study was undertaken to more carefully examine the non-5-HT1A/non-5-HTjg site in the rabbit caudate nucleus for comparison with the 5-HT1D site identified in the bovine caudate nucleus. 33

METHODS

Preparation of membranes for ligand-binding assays

The caudate nucleus (CN) was the brain region selected for examination in both the rabbit and bovine. This tissue has been well characterized as having a relatively high content of non-5-HT1A/non-5-HT1B binding sites in the rabbit (Schnellmann et al., 1984) and of 5-HT10 binding sites in the bovine (Heuring and Peroutka, 1987). The CN from fresh or frozen rabbit brains (from male New Zealand White rabbits) and frozen bovine brains (Pel

Freeze Biologicals) were removed and membranes prepared as previ ously described by Pedigo et al. (1981). In brief, the tissue was homogenized in 40 volumes of Tris-HCl buffer (50 mM, pH 7.4) using an~ :.•:maTJll polytron. This homogenate was centrifuged at 48,000 x g for 10 min. The pellet was resuspended and the process was repeated three more times. The membrane suspension was incubated at 37°C for 10 min between the second and third washes to facili tate removal of endogenous 5-HT (Nelson et al., 1978). The final pellet was resuspended in Tris buff1·r :50-100 ml/g of original tissue weight) for use in the assay.

[3H]5-HT binding assay--saturation studies

Saturation experiments were performed using [ 3H]5-HT in 34

the absence and presence of 100 nM 8-0H-DPAT and 100 nM mesulergine. Concentrations of [3H]5-HT ranging from 0.3 nM to 18 nM were

incubated with aliquots of a fixed concentration of tissue homogenate in order to determine total binding of the radioligand.

Nonspecific binding of [3H]5-HT at each radioligand concentration examined was determined in the presence of 10 uM 5-HT. Each set of determinations for both total and nonspecific binding were

performed in triplicate. Scatchard analysis of the data allowed the estimation of the number of binding sites per unit tissue

anc (Bmax) * the dissociation constants (K,j).

r3HT5-HT binding assay—inhibition studies

The binding of [3H]5-HT was determined as previously described by Bennett and Snyder (1976) with modifications (Nelson et al., 1978, Pedigo et al., 1981). In brief, the binding reac

tion medium consisted of 1 ml of resuspended membranes, 0.1 ml of

various concentrations of drugs, and 0.9 ml of [3H]5-HT (final concentration, 2 nM) in a buffer containing Tris-HCl, pargyline, ascorbate, 8-OH-DPAT and mesulergine to achieve final assay con centrations of 50 mM, 100 uM, 5.7 mM, 100 nM and 100 nM, respec tively, for a final pH of 7.4. Unless stated otherwise, a final concentration of 3 mM CaCl2 was also added to the reaction medium.

The mixtures were incubated for 10 min at 37°C, and the incubation was terminated by vacuum filtration through Whatman GF/B filters 35 followed by two 4-ml rinses with ice-cold 50 mM phosphate buffer.

Specific [3H]5-HT binding was defined as the difference between

[3H]5-HT bound in the presence and absence of 10 uM 5-HT.

F3Hl-5-HT binding assay—GTP and cation regulation

Caudate nucleus membranes from the rabbit and bovine were prepared as described above. The inhibition experiments by GTP were performed with or without the 3 mM Ca ? + after blockade of the

5-HTlA and 5-HTic sites by 100 nM 8-OH-DPAT and 100 nM mesulergine. The inhibition by 5-HT and saturation of [3H]5-HT were also performed in the absence and presence of 100 uM GTP with or with out 3 mM Ca2+ in the reaction medium. To test the ion specificity, NaCl, L1C1, KC1, MgCl2. CaCl2- or MnCl2 was included in the reaction mixture. All samples were assayed in triplicate.

All results were reported as specific binding, the difference between binding in the presence and absence 10 uM 5-HT. Percent change was reported as percentage of control binding.

|"3H15-HT binding assay—NEM regulation

After the preparation of tissues, the membranes were preincubated with varying concentrations of NEM in a total volume of 1.0 ml in Tris buffer containing pargyline (10 uM) with varying concentrations of NEM at 37° for 10 min. Radioactive ligands and competing drugs were then added for radiolignad binding as de 36

scribed above.

T3Hl5-HT binding assay—distribution of the

binding sites in rabbit brain

The caudate nucleus, hippocampus, frontal cortex, cere

bellum, cortex ventral to the rhinal salcus (CVRS), and midbrain

from fresh or frozen rabbit brains were removed and membranes

prepared as described above. A concentration of 2.0 nM [3H]5-HT

was used to define regional differences in total [3H]5-HT binding

versus [3H]5-HT binding in the presence of 100 nM 8-0H-DPAT and

100 nM mesulergine. Each experiment was repeated 3 times. Data

are described as the specific binding (fmol/mg tissue), the dif

ference between binding in the presence and absence 10 uM 5-HT.

Data Analyses

Binding parameters were determined by nonlinear regres

sion analysis (PCN0NL1N, Statistical Consultants, Inc.). For the

3 [ H]5-HT saturation isotherms the estimates of the Bmax and Kq

values were obtained by non-linear regression analysis of specifi

cally bound ligand as a function of ligand concentration. For the

inhibition studies of [3H]5-HT binding the estimates of overall

IC50 values were made graphically or by using the four parameter

logistic equation described by De Lean et al. (1978). In addi

tion, the inhibition curves were evaluated for fit to both 1-site 37 and/or 2-site models as described by Ehlert et al. (1980). The computer program PC NONLIN (Statistical Consultants. used, based on the following models:

one site: B a/(1 + x/K'H)

two site: B a/(1+x/K'H) + (1-a)/{1+x/K'L). where B is the proportion of [ 3H]5-HT bound, a and (1-a) are the proportions of high-affinity and low-affinity sites, respectively, and x is the concentration of the non-labeled drug. K'H and K'L are the apparent dissociation constants for the high- and low affinity binding sites, respectively.

The statistical method of DeLean et al. (1982) using a partial F-test was utilized to determine if the data would fit a two-site model significantly better than a one-site model. IC5o values were converted to apparent Ki values using the method described by Cheng and Prusoff {1973). Drug affinities determined in competition studies are expressed as pKi values (i.e., -log

Ki). Pearson correlation coefficients are reported as r values.

Significant differences between correlation coefficients was determined by the method of described by Bruning and Kintz (1987).

Differences in drug potencies were tested by use of analysis of variance, followed by the Newman-Keuls' post-hoc comparison to determine which means were significantly different. Statistical significance was considered when p< 0.05. 38

Materials

[3H]5-HT (30.4 Ci/mmole) was purchased from New England

Corp. (Boston, MA). Serotonin creatinine sulfate, pargyline,

GTP, N-ethylmaleimide (NEM), ergonovine, ergocryptine, ergotamine tartrate, and dihydroergotamine tartrate were purchased from Sigma

Chemical Co. (St. Louis, MO). Spiperone, spirilene, spiramide, and ketanserin tartrate were gifts from Janssen Pharmaceuticals

(Beerse, Belgium), and metergoline was a gift from Farmitalia

(Milan, Italy). Mesulergine, methysergide, dihydroergocristine, and dihydroergocryptine were gifts from Sandoz Research Institute

(East Hanover, NJ). 8-OH-DPAT (8-hydroxy-2-(di-n- propylamino)tetralin) was purchased from Research Biochemical Inc.

(Wayland, MA). TFMPP (l-(m-trifluoromethylphenyl)piperazine) was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Me- thiothepin and RU 24969 were gifts from Hoffmann-La Roche, Inc.,

(Nutley, NJ) and Roussel-UCLAF (Romainville, France), respective ly. Drugs were prepared fresh daily in distilled water or acetic acid and then diluted in distilled water before addition to reac tion mixtures. 39

RESULTS

Blockade of 5-HT^^ and 5-HT-]r> binding sites

in rabbit caudate nucleus

The initial step in the characterization of [3H]5-HT

binding sites in the rabbit caudate nucleus (CN) was to verify

that the conditions reported for masking 5-HT^A and 5-HTlc site9

in bovine CN could be used successfully in the rabbit.

As shown in Figure 1, competition curves with the

5-HT1A-selective drug 8-OH-DPAT and the 5-HTlc-selective drug

mesulergine against [3H]5-HT binding in the rabbit caudate were

best fit by a two-site model as determined by nonlinear regression

analysis. By this procedure it was estimated that 8-OH-DPAT

recognized 17.9 + 6.5 % of total specific [3H]5-HT binding sites

with high-affinity (K^ = 5.3 + 6.4 nM) and the remaining sites

with relatively low affinity (K^ = 848 + 211 nM). Similarly,

mesulergine recognized a low percentage (13.6 + 4.5 % ) of high-

affinity sites (Kj = 8.0 + 11.5 nM), and the remaining sites had relatively low affinity (Kj = 6016 + 1371 nM). This finding suggested that concentrations of 100 nM 8-OH-DPAT and 100 nM

mesulergine could be used to block the 5-HT1A and 5-HTjC sites without significantly affecting the remaining [3H]5-HT-labeled sites, as had been described for the bovine CN (Heuring and Per-

outka, 1987). Under this condition (i.e., pharmacologically

masking the 5-HT1A and 5-HTlc receptors) the competition curve for 40

a 1.0-1 ui a. m 0.8 ci 0.6-

0.4- 3 O CO 0.2- O—O No added DPAT & Meau.

in 0.0- •—• Added DPAT & Mesu. x" n -0.2 -r- ~1" -10 -9 -8 —7 -6 —5 -4 -3 LOG (Mesulergine), M

~ 1-2 o 1.0-1 Sui 0. v> 0.8 Q

"W£ 0.8 a § 0.4 1 A- -A No added DPAT A Mesu. « 0.0 A- Added DPAT Sc Mesu. I ro -0.2 1— , -10 -9 -8 -7 -6 -5 —3 LOG (8-OH-DPAT). M

Figure 1. 8-OH-DPAT and mesulergine competition studies

with [^H]5-HT binding in the absence and presence of 100

nM 8-OH-DPAT and 100 nM mesulergine in rabbit CN. In

creasing concentrations of 8-OH-DPAT and mesulergine

were incubated with 2.0 nM [^H]5-HT and rabbit CN as

described in Methods. Data shown are the results of

mean + SEM from 3 experiments. A. Competition curves of

mesulergine; B, Competition curves of 8-OH-DPAT. 41 mesulergine in the rabbit CN membranes could be adequately de scribed by a one-site binding model. In addition, the slope of the 8-OH-DPAT competition curve in the rabbit CN membranes was increased from 0.65 to 0.84, but it still fit a two-site model significantly better than a one-site model.

Saturation analysis of f3Hl5-HT binding in rabbit caudate nucleus

As noted above, a portion of the [3H]5-HT bound in rabbit CN was displaced by 8-OH-DPAT and mesulergine with nanomo

3 lar affinity, suggesting that [ H]5-HT labeled both 5-HTjA and

5-HTjq sites in this tissue. These two sites combined represented less than half of the total specific [3H]5-HT binding. Saturation experiments were, therefore, performed with [3H]5-HT both in the presence and absence of 100 nM 8-OH-DPAT and 100 nM mesulergine to characterize these sites. As shown in Figure 2 and Table 2, in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine, [3H]5-HT labeled with high affinity a saturable population of sites in membranes of rabbit CN. Non-linear regression analysis of the saturation curves indicated that L3H]5-HT labeled an apparently homogeneous population of high-affinity recognition sites (Kp =

4.14 + 0.48 nM). The proportion of non-5-HT1A/non-5-HTlc binding sites in rabbit CN (Bmax = 14.31 + 0.71 fmol/mg tissue) was about

11% of the total 5-HTj sites (Bmax = 18.57 + 1.26 fmol/mg tissue, determined in the absence of added 8-0H-DPAT or mesulergine). 42

•—• 3H-5-HT O—O 3H-5-HT + 100 nM DPAT ft MESULERGINE H- 4 8 8 10 12 14 16 18 20 pH]3—HT FREE, nM

O 3H—5—HT

SH-3—HT 4- 100 nM DPAT u. * MESULERGINE CD

4 8 8 10 12 14 16 18 20 B (fmoto/mg tissue)

Figure 2. [3H]5-HT saturation studies in rabbit CN in the ab sence and presence of 100 nM 8-0H-DPAT and 100 nM mesulergine. Increasing concentrations (0.3-18 nM) of [3H]5-HT were incubated with rabbit caudate membranes under standard assay conditions or in the presence of 100 nM 8-0H-DPAT and 100 nM mesulergine as described in Methods. Data shown are the results of mean from three experiments. Scatchard data were analyzed by linear- regression analysis. A, specific [3H]5-HT binding. B, Scatchard analysis of data shown in A. 43

Table 2. Results of saturation experiments performed in rabbit and

bovine CN with [3H]5-HT in the absence and presence of 100

nM 8-OH-DPAT and 100 nM mesulergine.

Tissue Binding Kfj(nM) Bmax % total

Rabbit CN Total 5,.25 + 0..96 18..57 + 1..26 100

•8-OH-DAPT & 4., 14 + 0,,48 14.,31 + 0.,71* 77 mesulergine

Bovine CN Total 6., 12 + 0..78 12..65 + 1,.56 .100

+8-OH-DPAT & 8..40 ov> .or.. 11 .. •'! fi •i 1 .42, 90.6 - mesulergine

Membranes (5-10 mg/assay) were incubated in the presence of

[3H]5-HT alone (total binding), or in the presence of 100 nM

8-0H-DPAT and 100 nM mesulergine (to mask 5-HTj^ and 5-HTjc

binding). Non-specific binding was determined in the presence of

10 uM 5-HT. Affinity values are expressed as values (nM) and

Bmax values as fmol/mg tissue + SEM of three separate experiments.

* p < 0.05, compared to total value in this tissue. 44

This agreed with the proportion of non-5-HTiA/non-5-HTlc sites

determined by the competition studies discussed above. However,

in the membranes of bovine CN, [3H]5-HT labeled approximately 10 %

fewer binding sites (Bmax = 11.46 + 1.42 fmol/mg tissue) than

under standard assay conditions (12.65 + 1.56 fmol/mg tissues).

This 10% decrease in Bmax was consistent with the data published

by Heuring and Peroutka (1987).

Comparison of the pharmacologic profiles of T3H15-HT binding

sites in rabbit and bovine caudate nucleus (CN)

after blockade of 5-HT^ and 5-HT|r sites

Comparison of the binding site in rabbit CN and 5-HTjA, 5-HTjg,

5-HTj0, and 5-HT2

To further characterize the [3H]5-HT binding sites in

rabbit CN, the pharmacologic profile was examined in the presence

of 100 nM 8-OH-DPAT and 100 nM mesulergine to mask the 5-HT^ and

5-HTjq sites. Figure 3 shows that two potent 5-HT-^-active drugs

were relatively weak in competing for [3H]5-HT binding in rabbit

CN. Although 8-OH-DPAT and spiperone displayed nanomolar affinity

for 5-HT1A sites in rat cortex (Peroutka, 1986), they competed for

specific [3H]5-HT binding in rabbit CN at micromolar concentra

tions .

By contrast, certain 5-HTjg-selective drugs were more

potent competitors of [3H]5-HT binding sites in rabbit caudate

(Fig. 4). Computer analysis of RU 24969 interactions with the 45

specific [3H]5-HT binding indicated that the data were adequately explained by a single population of binding sites with a Ki value of 70.8 + 1 nM. The TFMPP also dislpaced [3H]5-HT in a monophasic manner.

Drugs reported to display relatively high affinity for the 5-HT2Q binding site subtype (Pazos et a]., J984; Hoyer et al.,

1985) were markedly less potent in competing for [3H]5-HT binding in rabbit CN (Fig. 5). Thus, methysergide and mesulergine with both have affinity constants at the 5-HTjq site of about 2 nM, had

Kj values of approximately 152 nM and 3900 nM, respectively, at the [3H]5-HT binding sites in rabbit CN.

As shown in Fig. 6, the 5-HT2-selective drugs ketanserin and spiperone were relatively weak in competing for [3H]5-HT binding in rabbit CN , although they displayed nanomolar affinity for 5-HT2 sites in rat cortex (Leysen, 1981).

Table 3 and Fig. 7 shows that the correlations between the non-5-HTj^/non-5-HTic [3H]5-HT binding sites in rabbit CN and

5-HTia. 5-HT1b> 5-HTjq or 5-HT2 binding are much less significant or nonexistent.

As described above, the binding sites in rabbit CN sug gested that they did not correspond to at least several known 5-HT receptor subtypes based on their lower affinity for compounds with high affinity and/or selectivity for 5-HT1a (8-OH-DPAT, spiperone), 5-HTjg (RU 24969, TFMPP), 5-HTjq (mesulergine), and 46

1.2 O

O UJ 1.0-j Q- V) • 0.8-j o < ) a: u. 0.6 -j i o z 0.4-I :od m 0.21 A A SPIPERONE x «2, 0.0 A—A 8-OH-DPAT x ro -0.2 P 1 , , -10 -9 -8 -7 -6 -5 -4 -3 LOG (DRUG). M

Figure 3. Competition studies of 5-HTj^-selective drugs with the

non-5-HT1A/non-5-HTlc binding sites in rabbit CN.

Competition studies with [3H]5-HT (2.0 nM) binding in

the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine

were performed as described in Methods. Data are ex

pressed as the mean + S.E.M from three separate experi

ments, each point was carried out in triplicate. 47

1.2 O I g 1.0 LLJ OL 00 0.8 H 0 1 °-6i \ a ON 0.4 i\\ 3 O

f° 0.2-i O O RU 24969 0\ X I to 0.0-i TFMPP X ro -0.2 1 I j 10 -9 -8 -7 -6 -5 -4 -3 LOG (DRUG), M

Figure 4. Competition studies of 5-HTig-selective drugs with the

non-5-HTiA/non5_HTlc [3H]5-HT binding sites in rabbit

CN. Competition studies with [3H]5-HT (2.0 nM) binding

in the presence of 100 nM 8-0H-DPAT and 100 nM mesuler-

gine were performed as described in Methods. Data are

expressed as the mean + S.E.M. from three separate

experiments, each point was carried out in triplicate. 48

1.2 O Lju O 1.0 LxJ •l. in 0.8- o 0.6-

a z 0.4 3 O CD 0.2 O O METHYSERGIDE J— X I • • MESULERGINE 10 0.0-I I—I X ro —0.2 -10 -9 -8 -7 -6 -5 -4 -3 LOG (DRUG), M

Figure 5. Competition studies of 5-HTjC-selective drugs to the

3 non-5-HT1A/non-5-HTlc [ H]5-HT binding sites in rabbit

CN. Competition studies with [3H]5-HT (2.0 nM) binding

in the presence of 100 nM 8-0H-DPAT and 100 nM mesuler-

gine were performed as described in Methods. Data are

expressed as the mean + S.E.M. from three separate

experiments, each point was carried out in triplicate. 49

O tz o UJ £L 1.0- (/) Q 0.8 --

0.6- i

3 O 0.4- m h" x 0.2- O—O SPIPERONE\ I in • • KETANSERINE T I \ 0.0- i—ix 6 ro . 2- -10 -9 -8 -7 -6 -5 -3 LOG (DRUG). M

Figure 6. Competition studies of 5-HT2-selective drugs to the

non-5-HTjA/non-5-HT!Q 5-HT binding sites in rabbit CN.

Competition studies with [3H]5-HT (2.0 nM) binding in

the presence of 100 nM 8-0H-DPAT and 100 nM mesulergine

were porformed as described in Methods. Data are ex

pressed as the mean + S.E.M. from three separate experi

ments, each point was carried out in triplicate. 50

L pKj IN RABBIT CN IN RABBIT CN

pK| IN RAB8fr CN pK| IN RABBIT CN

O Figure 7. Correlation between the non-5-HTj^/non-5-HTjQ [ H]5-HT

binding site in the rabbit CN and 5-HTj^, 5-HTjg, 5-

HT1C, and 5-HT2 binding sites. pKp values for 5-HT1R

binding sites are from Table 4, pKjj values for the other

recognition sites are from Hoyer et al. (1985). 51

Table 3. Correlation between 5-HT1R binding and 5-HT1A, 5-HT1B,

5-HTlc. 5-HT jo , and 5-HT2 binding

Parameters 5-HT1A 5-HT1B 5-HTlc 5-HT1D 5-HT2

Slope 0.54 1.06 0.14 0.77 0.40

Intercept 3.66 0.89 6.44 5.10 10.24

Correlation 0.57 0.80 0.13 0.86 0.31 Coefficient

P value <0.05 <0.01 >0.05 <0.01 >0.05

n 11 11 11 11 11

The affinity profile of 5-HTjp sites determined in rabbit caudate membraines was compared to the affinity profiles of 5-HT1A, 5-

HTifi. 5-HTlc, 5-HTiq, or 5-HT2 sites determined as described

(Hoyer et al., 1985). 52

5-HT2 (spiperone) sites. The compounds having the highest

affinity for the rabbit CN sites were the indoles (5-HT and RU

24969) and the ergolines (raetergoline and methysergide).

Comparison of the binding site in rabbit CN and 5-HTjq in bovine

Table 4 shows the comparison of the affinity values of

various structurally and pharmacologically different compounds for

3 the high affinity non-5-HTj^/non-5-HTlc [ H]5-HT binding sites in

rabbit and bovine caudate membranes. Affinity values for the

compounds at the 5-HT^d site recently described by Heuring and

Peroutka (1987) in bovine brain are also included for comparison.

If spiperone and spirilene, an analog of spiperone, are

left out of the comparison, a significant correlation is obtained

between the pKj values in the rabbit and bovine CN (Fig.8A; r

0.8644, p <0.01). However, an even better correlation is ob

tained when comparing the pKj values in bovine CN obtained in the

present study with those reported by Heuring and Peroutka (1987)

for the 5-HTjjj sites (Fig. 8B; r = 0.9895, p < 0.001). In fact,

the correlation coefficient between the two sets of values for the

bovine CN from the two different laboratories was significantly

better than that for the comparison between the rabbit and bovine

CN carried out in our laboratory under exactly the same conditions

(p < 0.05). 53

Table 4.Affinity values of drugs for [3H]5-HT binding sites in

rabbit caudate nucleus (CN) in comparison to bovine brain

5-HTjq sites

DRUG pKj

a Rabbit CN Bovine CN 5-HT1D

8-0H-DPAT 6.,27 + .05 6,,58 + .11 6.2

RU 24969 7,, 16 + .17 7. CO .05 7.6 1 +

TFMPP 7,, 16 + . 16 6,.76 + .06 6.5

Mesulergine 5,.41 + .04 5,,72 + .22 5.2

5-HT 7..96 + . 10 8.,15 + .03 8.5

Methiothepin* 6,,77 + .14 7,,55 + .05 7.4 to Metergoline* 7..24 + .06 8, CD .04 8.4 1 + 00 Methysergide 6..92 + 7. co .19 6.9

.20 1+

• cr b Spiperone 5,,56 + . 13 A <4 cr

Spirilene 5,.47 + .07 A

Spiramide 5.,09 + .13 5.,14 + .07

Ketanserin 5..56 + .21 5,,73 + . 18 4.3

Affinity values of drugs competing for non-5-HTj^/non-5-HTiQ 5-HT binding sites labeled with 2.0 nM [3H]-5HT in rabbit and bovine CN are expressed as pKj values (-log Kj, M). Data represent the means + the S.E.M. from three to five separate experiments per formed in triplicate. The apparent Kj values were calculated using the Cheng-Prusoff equation (Cheng and Prusoff, 1973) using Kd values of 10 nM and 7 nM for [3H]5-HT binding sites in the rabbit and bovine CN, respectively. a Data taken from Heuring and Peroutka (1987) for the 5-HTjd binding site in bovine CN. Less than 50% inhibition of specific [3H]5-HT binding at a drug concen tration of 100 uM. * indicates a significant difference between the rabbit and bovine CN (p < 0.05). 54

Z CJ 8 SLOPE « 0.7693 t= T r =» 0.8644 m 5 p < 0.01 tr

tn Ui z> £ 6 + Q.

8 pKj VALUES IN BOVINE CN

B i zu Poo „8-r| SLOPE - 1.307 ... O) r - 0.989S p <0.01 m

c/i Ui 3

8 9

pK[ VALUES IN BOVINE CN

Figure 8. Correlations between the pKj values of compounds for the

3 non-5-HT1A/non-5-HTlc [ H]5-HT binding sites in rabbit and

bovine CN. The pKj values are taken from Table 4. A.

Correlation between pKj values in rabbit and bovine CN.

B. Correlation between pKj values in bovine CN and the

5-HTjd site (bovine CN, Heuring and Peroutka, 1987). 55

Effect of spiperone and analogues of spiperone on the 5-HT binding

sites in rabbit and bovine CN

Spiperone is a potent antagonist for both 5-HT2 and 5-

HTja receptors (Leysen, 1981; Pedigo et al., 1981). The effects

of spiperone and some analogs of spiperone on the [3H]5-HT binding

were examined in rabbit and bovine CN. From Table 4 and Figure 9,

it can be seen that a large difference in affinity of spiperone

and spirilene exists between the rabbit and bovine CN binding

sites. Thus, even at 100 uM spiperone and spirilene did not

produce 50% inhibition of [3H]5-HT binding in the bovine CN, while

in rabbit CN the apparent K^ values were 3.2 uM and 3.5 uM, re

spectively. The specificity of spiperone and spirilene was quite

striking since a closely related compound, spiramide (Fig. 10),

showed equal potency in both tissues. The affinity constant for

spiperone calculated in the presence of 8-OH-DPAT and mesulergine

to mask 5-HTjA and 5-HTlc sites was very similar to that previous

ly estimated by two-site analysis in rabbit CN in the absence of

masking drugs (2.4 uM; Schnellmann et al. 1984). As shown in

Figure 9 and Table 5, competition curves with the spiperone and

spirilene against [3H]5-HT binding in the bovine caudate were best

fitted by a two-site model as determined by nonlinear regression

analysis. Spiperone recognized 39.0 % of total specific [3H]5-HT

binding sites with high-affinity (Kj = 2242.5 + 677.5 nM) and the

remaining sites with relatively low affinity (Kj >= 100,000 nM).

Similarly, spirilene recognized 38.7% of high-affinity sites (Kj 56

1.2-r O GZ o UJ 1.0-- 0- V) d < 0.8 -- a 0.6 -- 2 !D A A SPIPERONE, RON O ffl 0.4-- • A SPIPERONE, BCN O O SPIRILENE, RCN m 0.2-- i—i •—•SPIRILENE, BCN x ro 0.0 + -10 -9 -8 -7 —6 -3 LOG [DRUG], M

Figure 9. Inhibition curves for spiperone and spirilene against

non-5-HT1A/non-5-HTlc [3H]5-HT binding in rabbit and

bovine CN. Open symbols represent the rabbit CN and

closed symbols the bovine CN. Triangles are for spiper

one and circles for spirilene. Each point represents

the mean + S.E.M. for three separate experiments, each

carried out in triplicate. 57

p~0~^L ° SPIPERONE

NH SPIRILENE

N SPIRFmiDE:

Figure 10. Chemical structures of spiperone and its analogs. 58

Table 5. Computer-assisted nonlinear regression analysis of

spiperone, spirilene and splramide interactions with

[3H]5-HT binding in rabbit and bovine CN.

Rabbit CN Bovine CN Compounds

Ki(nM) K1 K;

Spiperone 6943.2 + 785.6 2242.5 + 611.7 >100,000

Spirilene 3469.4 + 601.8 1011.0 +1314.5 >100,000

Spiramide 8704.2 + 2239.5 7394.4 + 1087.6

Competition studies were performed as described in Methods using 2 nM [aH]5-HT. Computer-assisted analysis of the competition curves was performed according to the method of and w:is used to dcti-i mine Kj values and relative concentrations of each binding site.

Each curve was fitted according to both a one-site and a two-site model. 59

1011.04 nM) and the remaining sites had relatively low affinity

(K 1 >= 100,000 nM). This finding suggested lhdl 1D ; (~ bovine CN might be hetergenous. Comparison of the pKi values obtained in the two tissues examined in this study also showed that the affinities for metergoline, methiothepin and ergonovine were statistically significantly different between two tissues.

Effect of some ergots on the 5-HT binding sites in both tissues

The effects of some ergot compounds on the 5-HT binding were examined in rabbit and bovine CN. As shown in Table 6, all the ergots and dihydroergots shown high affinity for the 5-HT binding sites in rabbit and bovine CN. In addition, a significant difference in the affinity values of ergonovine existed between rabbit and bovine CN (Fig. 11). Competition curves with the ergotamine and ergocryptine against [3H]5-HT binding both in the rabbit and bovine CN were best fitted by a two-site model as determined by nonlinear regression analysis (Fig. 12). It can be seen from Table 6, in the rabbit CN, that ergotamine and ergo cryptine recognized respectively 60% and 39% of total specific non-5-HT1A/non5-HT1c 5-HT binding with high affinity (Ki 0.014 and 0.015 nM) and the remaining sites with relatively low affinity

( Ki = 144.5 and 74.5 nM), respectively. In the bovine CN, ergo tamine and ergocryptine recognized respectively 68.1% and 72.2% of the sites with high-affinity sites (Ki = 0.05 and 0.18 nM) and

the remaining sites with relatively low affinity (Ki = 97.6 and 60

"1.2-|— — — .... O C o 1 .o 4- Ul Q. I o O O RCN (/) 0.8 + \ • ' • • BCN i \ 0.6 j 9 a i z -j- z> 0.4 \ 0 l • m j »- 0.2 f x i 1 j in 0.0 -*• i—ix to -0.2 -10 -9 -8 -7 -6 -5 -4 -3 LOG (DRUG), M

Figure 11. Inhibition curves for ergonovine against non-5-

3 HTlA/non-5~HTlC [ H]5-HT binding in rabbit and bovine

CN. Each point represents the mean + S.E.M. for three

separate experiments, each carried out in triplicate. 61

~ 1.2-1— y i 1 g 1.0- ^ ° O ERGOCRYPTINE, RCN gj •- "• ERGOCRYPTINE, BCN 0.8 o < QL 0.61 \ a z ZD 0.4 4- o m 0.24- i | A A ERGOTAMINE, RCN < * ^wv Q 0-0 ± A- -A ERGOTAMINE, BCN ro • 1—1 -0.2 4 1 1 1 1 f— -14 -12 -10 -8 -6 -4 LOG (DRUG), M

Figure 12. Inhibition curves for ergotamine and ergocryptine

against non-5-HTj^/non-5-HTiQ [%]5-HT binding in rabbit

and bovine CN. Each point represents the mean + S.E.M.

for three separate experiments, each carried out in

triplicate. 62

s 1.0"

0.6-•

0.4+

O RCN • BCN !0.0-•

-14 -12-12 -10 -8 -e -4 LOO (HHVDROERQOTAMME), M

O RCN • BCN

-12 -10 -8 -« LOG (DtHYDROEWOOCWSTINE). M

* 0.2-• • BCN

-12 -10 -8 -6 loo (owvmwoioocmpnNg). u Figure 13. Inhibition curves for dlhydroergotamlne, dihydroergocristine, and dihydroergocryptine against the non-5- 3 HTiA/non-5-HTjc [ H]5-HT binding in rabbit and bovine CN. Each point represents the mean + S.E.M. for three separate experiments, each carried out in triplicate. A: Inhibition curves of dlhydroergotamlne; B: inhibition curves of dihydroergocristine; C: Inhibition curves of dihydroergocryptine. 63

Table 6. Computer-assisted non-linear regression analysis of

competition curves of some ergots and dihydroergots with

non-5-HT1A/non-5-HT1c binding in rabbit and bovine CN.

Rabbit CN Bovine CN Compounds K1 K2 K1 K2

Ergotamine 0.01tl ] iJ.l . b 0. ii5 9'i

Ergocryptine 0.015 74.5 0.18 713.3

Ergonovine 20.84 3.42

Dihydroergotamine 0.41 1110.1 8.9

Dihydroergocristine 25.75 45038.5 90.2

Dihydroergocryptine 1.74 3083.5 83.47

Competition stud.ies were pcrforu . ; dr.scribed jn Metl1ods using

2.0 nM [ 3H]5-HT. Computer-assisted analysis of the competition

curves was performed according to computer program NONLIN and was used to determine Ki values (nM) and relative concentrations of each binding site. Each curve was fitted according to both a one-site and a two-site model. 64

713.3 nM). In contrast to the ergots discusscd above, the

competition curves with the dihydroergots were different in the

rabbit and bovine CN (Fig. 13). In the bovine CN, they were best

fitted by a one-site model (Table 6, Fig. 13), while they were

best fitted by a two-site model in the rabbit CN. These findings

suggested that ergot compounds have high affinity for the [3H]5-HT

binding sites in rabbit and bovine CN, the non-5-HTj^/non-5-HTic

[3H]5-HT binding sites in rabbit and bovine CN might acturally

represent a heterogenous population of binding sites, which is

consistent with the inhibition curves of spiperone and spirilene

in bovine CN.

Effect of GTP on T3H15-HT binding

to rabbit and bovine caudate nucleus membranes

Since the 5-HTjq sites of bovine CN, as well as other

5-HT binding sites, have been shown to be sensitive to guanine

nucleotides, the effects of GTP on specific [3H]5-HT binding in

the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine were

examined in the rabbit CN for comparison with the bovine CN. As shown in Fig.14, GTP significantly decreased specific [3H]5-HT

binding in rabbit and bovine caudate nucleus. The potency of the

inhibition was significantly less in rabbit caudate nucleus than

that in bovine caudate nucleus.

The effects of GTP on agonist inhibition of [3H]5-HT

binding were examined in rabbit and bovine CN. As shown in Fig. 65

O LL. o 1 -0 4- UJ ou cn o 0.8-- <

0.6-

3 m 0.4-- h- X O— OBCN, NoCa2+ in 0.2 + •—i 2+ x •—•RCN, No Ca to 0.0 -9 -8 -7 -6 -5 -4 -2 Log [GTP], M

3 Figure 14. Effects of GTP on the non-5-HT1A/non-5-HTlc [ H]5 HT

binding in rabbit and bovine CN. Each point (mean +

S.E.M. of the values from three separate assays, each

carried out in triplicate) is expressed as the fraction

of specific binding relative to the appropriate control

values, i.e., in the absence of GTP. 66

1,2T E 1.0- BOVINE CN. NO Cq2+ % 0.8- • O ONO GTP |0.6 - n • •+GTP z 0.4 - • § m 0.2-- ?E P-IJ, 0.0 • z IO -0.2 -10 -9 -8 -7 -6 -3

LOG [5—HT], M

1.2 a E 1.0 B S RABBIT CN. NO Ca2+ 8i 0.8

0.6 O ONO GTP a • +GTP z 0.4 3 s 0.2

r—iio 0.0 • • X IO ,-J -0.2 -4- -10 -9 -8 -7 -6 -5 -4 -3

LOG [5—HT], M

Figure 15. Effect of GTP on agonist inhibition of [ H]5-HT bind

ing to non-5-HT!^/non-5-HTic sites in rabbit and bovine

CN. 5-HT inhibition of [3H]5-HT (2.0 nM) binding (in

the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine)

was carried out with (#) or without(O) the addition of

100 uM GTP. A.in the bovine CN; B.in the rabbit CN. 67

O NO GTP + GTP £ 10 12 14 16 18 20 [3H]5-HT FREE. nM

B O NO GTP • + GTP

6 8 10 12 14 16 18 B (FMOLES/MG TISSUE)

Figure 16. [3H]5-HT saturation studies in the presence and absence

of 100 uM GTP in rabbit CN without calcium. Increasing

concentrations (0.3-18 nM) of [^H]5-HT were incubated

with rabbit or bovine CN membranes in the presence of

100 nM 8-OH-DPAT and 100 nM mesulergine as described in

Methods. Data are expressed as the mean from three

separate experiments. Scatchard data were analyzed by

linear-regression analysis. 68 18T 16 j* a A z o 14 o O- -O NO GTP m o 12-- -• + GTP C a 10-- ui a. CO 8--

J 6 in i—i x 4-- rj 2--

1- —h H 1 1 h 6 8 10 12 14 16 18 20 [3H]5-HT FREE, nM

B O NO GTP • + GTP

8 12 16 20 24 B (FMOLES/MG TISSUE)

Figure 17. [3H]5-HT saturation studies in the presence and absence

of 100 uM GTP in bovine CN without calcium. Increasing

concentrations (0.3-18 nM) of [®H]5-HT were incubated

with rabbit or bovine CN membranes in the presence of

100 nM 8-0H-DPAT and 100 nM mesulergine as described in

Methods. Data are expressed as the mean from three sepa

rate experiments. Scatchard data were analyzed by

linear-regression analysis. 69

Table 7. Results of saturation experiments performed in the rabbit

and bovine CN to the non-5-HTi^/non-5-HTiQ [3H]5-HT bind

ing in the presence and absence of 100 uM GTP without

calcium

Tissue Binding Bmax

Rabbit No GTP 7.88 + .96 16.39 + 2 .80

+ GTP 8.44 + 1..79 14.54 + . 41

Bovine No GTP 16.2 + 4.,07 25.33 + 5 .02

+ GTP 37.0 + 2.,90* 23.02 + .43

Membranes (5-10 mg/assay) were incubated in the presence of various concentrations of [®H]5-HT, or in the presence of 100 uM

GTP. Non-specific binding was determined in the presence of 10 uM

5-HT. Affinity values are expressed as Kj values (nM) and Bmax values as fmol/mg tissue + S.E.M. of three separate experiments.

* p < 0.02, compared to the values without GTP in this tissue. 70

1[), .ir1 ttw h)\ ine CN GTP at a concentration of 100 uM significant ly shifted the 5-HT binding to the right in the absence of calcium

(p < 0.05; Fig. 15A). In contrast, in the rabbit CN, GTP at the same concentration did not modulate 5-HT binding in the absence of calcium (p > 0.05; Fig. 15B). These findings suggested that there are different GTP effects on the affinity values (Kd) of

[3H]5-HT binding between rabbit and bovine CN with more potent GTP effects on the bovine CN, which was consistent with the above results of GTP modulation in both tissues.

In order to further characterize the GTP effect on the binding of 5-HT, the effects of GTP on the saturation of [ 3H)5-HT binding were also examined in rabbit and bovine CN. As shown in

Fig. 16 and 17 and in Table 7, in the rabbit CN, GTP at concen tration of 100 uM almost had no significant effect on either the affinity value (Kd) or Bmax of [ 3H]5-HT binding, while in the bovine CN, GTP at same concentration significantly shifted the affinity values from 16.2 nM to 37 nM (Table 7. ). These findings suggested that in the absence of calcium, GTP had no effect on

Bmax in either the rabbit or bovine CN. However, in combined with tlw results from the C ~h.ift studies, it suggested that GTP, without calcium, could influence the affinity of [ 3H]5-HT in bovine CN and had no effect on the affinity in rabbit CN.

Interaction of GTP and cations on [3H]5-HT binding

Since the 5-HTlD sites of bovine CN, as well as other 5- 71

HT binding sites, have been shown to be sensitive not only to guanine nucleotides but also to divalent cations, the interaction of GTP and calcium with specific [3H]5-HT binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine was examined in the rabbit CN for comparison with the bovine CN. As shown in Fig. 18, the addition of 3 mM calcium caused a decrease in total binding O and a decrease in nonspecific binding of [ H]5-HT to the rabbit

CN homogenates. The total binding was decreased by 9.0 + 3 %, and the nonspecific binding was reduced by 16.9 + 5 % in the presence of 3 mM CaCl2- The net result was almost no change in the specif ic binding of [3H]5-HT to rabbit CN membranes (see Fig. 18 legend). The presence of 3 mM calcium in the reaction medium significantly increased the sensitivity of the binding of [3H]5-HT to inhibition by GTP in the CN of rabbits (Fig. 18A), i.e., it shifted the inhibition curve to the left. In the bovine CN, the addition of 3 mM calcium to the binding assay caused an increase in total binding by 5.2 + 3.0 % and a decrease in nonspecific binding by 12.0 + 3.5 %, yielding an increase in specific [3H]5-HT binding of 39.4 +81 In contrast to the rabbit CN, the sensi tivity of [3H]5-HT binding to GTP in the bovine CN was signifi cantly decreased in the presence of calcium (Fig. 18B), a phenome non which has been described previously by Heuring and Peroutka

(1987) for the 5-HT1D binding sites. 72

o E a 1.0- ui OL cn RABBIT CN 0.8-

0.6- a z 13 m 0.4- £ •—• no Ca2+ 0.2- i—i O— O + Co2+. 3 mM i in 0.0- -9 -8 -7 -6 -5 -4 -3 -2

Log [GTP], M / 1 u o —

E / a 1.0 B ui a. to BOVINE CN 0.8 1

0.6 1 a \ z 3 O s CD 0.4 #—9 no Co2+ in 0.2-- O—0+Ca2+.3mM X n 0.0 1 1 -1 1- » ' -9 -8 -7 -6 -5 -4 -3 -2 Log [GTP], M

Figure 18. Effects of GTP on the binding of [3H]5-HT in the

presence of 100 nM 8-OH-DPAT and 100 nM mesulergine with

(O) and without (#) 3 mM calcium. A: in rabbit caud

ate membranes; B: in bovine caudate membranes. Each

point (mean + S.E.M. of the values from three separate

assays, each carried out in triplicate) is expressed as

the fraction of specific binding relative to the appro

priate control values, i.e., in the absence of GTP. 73

The effects of GTP and calcium on agonist inhibition of

[3H]5-HT binding were also examined in rabbit and bovine CN. As shown in Fig. 19, calcium at a concentration of 3 mM inhibited the ability of GTP to modulate 5-HT binding in the bovine CN (p <

0.05, Fig. 19A). In contrast, in the rabbit CN, calcium at same concentration significantly increased the shift of the 5-HT inhi bition curve to the right by GTP, increased the sensitivity of

GTP modulation to the binding.

We also examined the effects of GTP on the saturation of

[3H]5-HT binding in rabbit and bovine CN in the presence of 3 mM calcium. As shown in Fig. 20, 21, and Table 8, in the rabbit CN,

GTP at concentration of 100 uM significantly decreased the affini ty values without effect on Bmax, while in the bovine CN, GTP had no significant effect on either the affinity values or Bmax values. This is in agreement with the results from the G shift experiments in the presence of calcium.

To test the ion specificity of the effect of calcium on

GTP regulation of [%]5-HT binding, the effects of various monova lent and divalent cations on [3H]5-HT binding were examined in the presence and absence of 100 uM GTP in both tissues. Fig. 22 O summarizes the effects of cations on the [ H]5-HT binding in both tissues without the GTP. The divalent cations (e.g., Mn2+, Mg2+,

Ca2+) increased the specific binding, while monovalent cations decreased the specific binding in both tissues. There was a significant difference of Ca2+ effect on both tissues. Ca2+ 74

oE 1.0+-UT Ul QL W 0.8 + BOVINE CN. +Cq2+

0.6-- O ONO GTP • •+GTP z 0.4 + 3 O 03 0.2--

r—im 0.0 - • I r> -0.2 -10 -8 -7 -6 -5 -4 —3

LOG [5-HT], M

1.2 0 B § 1.0 ui RABBIT CN. +Ca2+ a. vt 0.8 6 ONO GTP 1 °--- • +GTP | 0.4

^ 0.2 £ i—iin 0.0 tox -0.2 -10 -9 -8 -7 -6 -5 -4 -3

LOG [5-HT], M

Figure 19.Effect of GTP on agonist inhibition of [3H]5-HT bind

ing to non-5-HTjA/non-5-HT1Q sites in rabbit and bovine

CN in the presence of 3 mM CaC^- 5-HT inhibition of

[3H]5-HT (2 nM) binding (in the presence of 100 nM

8-OH-DPAT and 100 nM mesulergine) was carried out with (4

) or without (O) the addition of 100 uM GTP. A. Modu

lation of 5-HT binding by GTP in the bovine caudate. B.

Modulation of 5-HT binding by GTP in the rabbit caudate. 75

B I '

8 10 12 14 18

B (tmole8/mt tlaue)

Figure 20. [ 3H]5-HT saturation studies in the presence and absence of 100 uM GTP in the rabbit with 3 mM calcium. Increasing concentrations 3 {0 ~-18 nMl of [ H]5-HT were incubated with rabbit or bovine CN membranes in the presence of 3 mM calcium as described in Methods. Data are expressed as the mean from three separate experiments. Scatchard data were analyzed by linear-

regression analysis. 76

O—O NOCTP

+ GTP 0 E

1(A IE r—lJL

10 12 14 18 1B [^HJS-HT FREE. nU

O NOCTP 2+ Q + GTP O OO

«b

2 4 6 8 10 12

B (fmelw/mg Unut)

Figure 21. [3H]5-HT saturation studies in the presence and absence

of 100 uM GTP in the bovine CN with 3 mM calcium.

Increasing concentrations (0.3-18 nM) of [3H]5-HT were

incubated with rabbit or bovine CN membranes in the

presence of 3 mM calcium as described in Methods. Data

are expressed as the mean from three separate experi

ments. Scatchard data were analyzed by linear-

regression analysis. 77

Table 8. Results of saturation experiments performed in the rabbit

and bovine CN to the non-5-HTiA/non-5-HTic [3H]5-HT bind

ing in the presence and absence of 100 uM GTP with 3 mM

calcium

Tissue Binding Kj B max

Rabbit No GTP 4.14+0.48 14.31+0.71

+ GTP 11.80 + 2.4* 11.25 + 1.24

Bovine No GTP 5.34+3.06 11.46+1.42

+ GTP 14.77+5.11 8.23+0.56

Membranes (5-10 mg/assay) were incubated in the presence of var ious concentrations of [3H]5-HT, or in the presence of 100 uM GTP.

Non-specific binding was determined in the presence of 10 uM 5-HT.

Affinity values were expressed as Kj values (nM) and Bmax values as fmol/mg tissue + S.E.M. of three separate experiments.

* p < 0.05, compared to the values without GTP in this tissue. 78

O OH •BCN

o o & o 2 a z aa o c o Ui a. m i— x m i—i x ro Mn2+ Mg2+ Ca2+ Na+ K+ Li+

Figure 22. Cation effects on the non-5-HTjA/non_5_HTlC t H]5-HT

binding sites in rabbit and bovine CN membranes. Values

given were the mean + S.E.M. from three saparate

experiments, each was carried out in triplicate. Data

are given as the percent of specific binding using

2.0 nM [3H]5-HT, where 100* =2.45 fmol/mg wet weight

tissue. 79

Table 9. Effects of cations on regulation of [3H]5-HT binding by

GTP

% Inhibition of Specific Binding by 100 uM GTP Cations Rabbit CN Bovine CN

35 mM NaCl 20,.8 •+* 1,.8 52,. 1 + 2,.0

35 mM LiCl 19,.7 + 0 ,5 45.,5 + 1 . 1

35 mM KC1 29 .4 + 0.8 41,.4 + 1 .0

3 mM MgCl2 37,.2 + 0,.6* 38..2 + 2,. 5*

3 mM CaCl2 39 .7 0,,7* 38,.9 + 3 .2*

3 mM MnCl2 41 ,6, + 0,,9* 28.,6 + 3,.5*

Tris 21,.4 + 0,.6 56.,4 + 1 .2

Rabbit abd bovine braine membranes were incubated at 37 0 for 10 min with 2.0 nM [%]5-HT and the indicated concentration of cation in the presence of 100 uM GTP. Data were experssed as mean per cent inhibition by GTP + S.E.M.. All experiments were performed in triplicate and replicated at least three times.

* indicates a significant difference between the control values

(GTP in Tris buffer) and the percentage inhibtion by GTP + ca tions. 80

increased specific binding in bovine CN, however, it had no effect

on rabbit CN.

Table 9 summarizes the effects of cations on the [3H]5-HT

binding in the presence of 100 uM GTP in both tissues. Divalent

cations (e.g., Ca2+, Mg2 + , Mn2 + ) significantly decreased the GTP O inhibition on [ H]5-HT binding in bovine CN in comparison with

the inhibition in the presence of 100 uM GTP alone. Conversely,

divalent cations increased the GTP inhibition of [3H]5-HT binding

in rabbit CN. Thus, both Mg2+ and Mn2 + were able to mimic the

effect of calcium on GTP regulation of [3H]5-HT binding in both

tissues. The monovalent cations (e.g., Na+, Li+, K+) were more

potent than the divalent cations as inhibitors of [3H]5-HT binding

(as shown in Fig. 20). However, the presence of monovalent cation

with 100 uM GTP did not produce any significant change in [3H]5-HT

binding beyond that obtained in the presence of the GTP alone.

Thus, although the monovalent cations by themselves were more

potent inhibitors of [3H]5-HT binding than the divalent cations,

only the divalent cations affected GTP inhibition of [3H]5-HT

binding.

The effects of NEM on the T3H15-HT binding

in the rabbit and bovine CN.

Addition of N-ethymeiamide (NEM), an alkylating agent of

sulfhydryl group, to bovine or rabbit membranes resulted in a

reduction of [3H]5-HT binding to high affinity [3H]5-HT binding 81

sites. Fig. 23 shows the results of experiments in which mem

branes were preincubated with vavious concentrations of NEM at

37°C for 10 min before addition of 2 nM [3H]5-HT. The binding sites of both tissues were sensitive to NEM. However, there was a significant difference in NEM sensitivity between the two tissues.

Only about 40 % of binding sites in rabbit were sensitive to NEM, while almost all the binding sites in bovine were sensitive to

NEM.

Distribution of the non-S-HT-^/non-S-HTjc

binding sites in rabbit brain

A concentration of 2.0 nM [3H]5-HT was used to define regional differences in total [3H]5-HT binding versus [3H]5-HT

binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine. As noted in previous studies (Peroutka and Snyder, 1981), total [3H]5-HT binding was highest in the caudate, hippocampus, cortex ventral to the rhinal salcus (CVRS) (Fig. 24, and Table

10). However, a distinct difference existed between regions in

the amount of specific [3H]5-HT binding that remained in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine. In the caudate, approximately 75% of specific [3H]5-HT binding remained under this condition. In contrast, the majority of specific

[3H]5-HT binding in hippocampus was displaced by 100 nM 8-OH-DPAT and 100 nM mesulergine, with only 46 % of the original amount of 82 specific [3H]5-HT binding remaining . In cortex, the amount of specific binding remaining in the presence of 100 nM 8-OH-DPAT and

100 nM mesulergine ranged from 55* to 65%. While the midbrain and cerebellum contained relatively low total [3H]5-HT binding, they were similar in that they contained a very high percentage of sites not displaced by 100 nM 8-OH-DPAT and 100 nM mesulergine. 83

°Q, 1I 2

8 1.0+

I 0.8 + o z m 0.6-- o O- -OBCN, No Cq2+ Ll. 0 0.4 + LLJ RCN, No Cq2+ Q. 01 i- 0.2 X I m _ _ 57 0.0 + to -10 -9 -8 -7 -6 -5 -2 LOG (NEM), M

Figure 23. NEM sensitivity of the 5-HT binding site subtypes in

bovine and rabbit CN. Membranes were incubated with

various concentrations of NEM for 10 min at 37° before

addition of radiolabeled ligand and masking agents. 84

IO I CZ1 JH-5-HT 1 OS 3H-5-HT + 100 nM DRUGS CS z 8 T 3 i o 7| m I 1 o C o LlI 5 Q. T c/) 4 4- h- X to 3-- 1—1 X fO 2-- 1 0 M HIPP. CERE. CTX. CVRS. MD CN

Figure 24. Regional distribution of specific [3H]5-HT binding in

the absence or presence of 100 nM 8-OH-DPAT and 100 nM

mesulergine in rabbit brains membranes. Data are ex

pressed as the specific binding (fmol/mg wet weight

tissue) using 2.0 nM [^H]5-HT. 85

Table 10. Regional distribution of specific [3H]5-HT binding in

the absence or presence of 100 nM 8-OH-DPAT and 100 nM

mesulergine

Region [3H]5-HT [3H]5-HT + DPAT % of total + mesulergine binding

Caudate 7,,78 + 1 ., 16 5,,66 + 1 ,41, 72,,8

Hippocampus 8,,76 -L 0,,82 4.,04 + 0,,51 46,,2

Cerebellum 1,,06 4- 0,,22 0,,86 + 0,,35 81 ,0

Frontal cortex 4.,74 + 0.,55 2,,61 + 0,,49 55,, 1

CVRS 6.,56 + 0.,68 4,,07 + 0,,37 62,,0

Midbrain 4,,71 + 0,.08 3,,07 + 0,,49 65,.1

Binding assays were performed as described in Methods. Values given are the mean + S.E.M. of three assays. Data are expressed as the specific binding (fmol/mg tissue) using 2.0 nM [3H]5-HT. 86

DISCUSSION

The major findings of this study are: 1)[3H]5-HT labels a binding site in rabbit CN that is pharmacologically different from the 5-HT1A, 5-HT1B, 5-HT-jq, and 5-HT2 sites, and although there are strong similarities between this site and the 5-HTjd site in bovine CN, the rabbit CN binding site is also pharmacolog ically distinct from the 5-HTjd site; (2) the guanine nucleotide O effects on agonist affinity for [ H]5-HT binding in rabbit CN are markedly different than the GTP effects on the 5-HTjq site in bovine CN; (3) the interactions of GTP and divalent cations, i.e., calcium, in altering [^HJS-HT binding in rabbit CN is different from those previously described for 5-HTj sites; (4) the sensitiv ity to the inactivation by NEM in rabbit CN is different from that for the site in 5-HTjq in bovine CN; and (5) both 5-HTjq in the bovine CN and the novel binding site in the rabbit CN may be heterogeneous.

Under conditions where 5-HT1A and 5-HTjc receptors were pharmacologically masked, [3H]5-HT labels a population of sites in rabbit CN membranes, which were saturable, high-affinity, and homogeneous. From Table 4 and Fig. 9, it can be seen that this site has a pharmacological profile that is distinct from the

previously identified 5-HT1A, 5-HT1B, 5-HTlc, and 5-HT2 receptors, but similar to the 5-HTjj) site (Fig. 8), a novel 5-HT binding site 87

originally described in bovine CN (Heuring and Peroutka, 1987).

Pharmacological profiles similar to that of the 5-HT-jp site have now also been reported for a number of other species, such as, pig, rat, and human (Waeber et al., 1988). However, while similar to the 5-HTjq sites in bovine CN, the non-S-HT-^/non-S-HTjQ

[3H]5-HT binding site in rabbit CN (hereafter designated 5-HTjpj for brevity's sake) is pharmacologically distinct from the 5-HTjq site. The distinction of the 5-HT^p site was shown most clearly with spiperone and spirilene, which exhibited values of 3.2 uM and 3.5 uM, respectively, at the 5-HT1R site, but which produced less than 50% inhibition of [3H]5-HT binding to the 5-HTjd site even at concentrations as high as 100 uM. These compounds also serve to demonstrate the fine degree of structural specificity for distinguishing between the 5-HT1D and 5-HT1R sites, since spiramide, which is structurally very similar to spirilene and spiper one, does not discriminate between these two sites.

There are several possibilities that might account for the differences in the pharmacological profiles observed between the 5-HTjj) and 5-HTjr sites. First, they may represent identical proteins that are coupled to different systems in the two tissues, resulting in slight conformational differences. Alternatively, the 5-HTir site may be the rabbit's version of the 5-HTjj) site, i.e., a species specific [3H]5-HT binding site. A precedent for species differences in 5-HT-j sites has already been established, 88 i.e., the 5-HT-tg site appears to occur only in the rat and mouse

(Heuring et al., 1986). Finally, the 5-HTjp site may represent yet another 5-HT-j binding site subtype.

While the pharmacologic findings by i ' .n,. lvos suggest that the 5-HTjp and 5-HT^r sites may be distinct entities, the GTP effects on [°H]5-HT binding further reinforce this possibility.

For example, in the absence of added calcium, GTP produced no significant shift in the agonist inhibition curves in rabbit CN, but the converse was true in the bovine CN (Fig.15), where GTP significantly shifted the inhibition curves. This was in agree ment with the finding that GTP was much less potent in inhibiting

[^H]5-HT binding to the 5-HTjr site than to the 5-HTjq site

(Fig.14). Guanyl nucleotides have been shown to modulate the binding of radioligands to receptors coupled with GTP binding proteins (Hoffman and Lefkowitz, 1980; Maguire et al., 1983).

Agonist radioligands preferentially label the agonist high-affini ty state of the receptor presumably formed by the receptor/G- protein complex (Maguire et al., 1976; DeLean et al., 1980).

Guanyl nucleotides are thought to facilitate the dissociation of the receptor/G-protein complex (forming the low-affinity state), resulting in a decrease in agonist radioligand binding. There fore, our findings could indicate species differences in the structures of these two [%]5-HT binding sites resulting in dif ferences in their abilities to effectively interact with or acti vate G-proteins. 89

The difference between the bovine and the rabbit binding sites was further emphasized by the influence of calcium on the effects of GTP. Studies have shown that divalent cations, such as calcium, exert an effect on agonist binding opposite to that of

GTP on the 5 HTj binding sites measured in rat brain (Mallat and

Hamon, 1982; Blurton and Wood, 1986), the 5-HT1A site in rat hippocampus (Hall et al. , .1985), and the 5-HT1D site measured in

bovine CN (Heuring and Peroutka, 1987). In these previous studies calcium decreased the potency of GTP for inhibiting agonist bind

ing. In contrast, at the 5-HTjr site calcium appeared to have a permissive or facilitative effect on the action of GTP. Thus, in rabbit CN calcium increased the potency of GTP for inhibiting

[3H]5-HT binding, while in the bovine CN it decreased GTP's poten cy (Fig. 18). Similarly, in the absence of added calcium GTP had no effect on the 5-HT inhibition curve of [3H]5-HT binding to the

5-HT1R sites, but in the presence of calcium GTP was able to shift the curve to the right (Fig. 19). This was in contrast to the

5-HT1D sites where calcium inhibited the GTP-induced shift in the agonist inhibition curve.

The precise mechanism behind the permissive effect of calcium and other divalent cations on the GTP-induced inhibition of 5-HT binding to the 5-HT1R site is not known. If the 5-HTjr site is actually a serotonin receptor subtype, there are multiple possibilities for what may be taking place. Several explanations 90 are as follows: 1) calcium and other divalent cations may be altering the 5-HTj^ site to change its binding to the G-protein;

2) calcium and other divalent cations may be altering the G-pro tein, or changing its ability to interact with either GTP or the receptor; 3) calcium and other divalent cations may be changing the coupling of the effector to the receptor/G-protein system.

Alternatively, the effects of calcium may indicate that the G- protein coupled to the 5-HTjr site is quite different from the G protein which is coupled to the 5-HTjq site.

The different sensitivities of the binding sites to the sulfhydryl group alkylator NEM provided other evidence for the difference between these two tissues. NEM inactivated binding of

[^H]5-HT to high affinity binding sites in rabbit and bovine CN membranes to differing extents. Almost 100 X of the specific binding to 5-HTjq in bovine CN were sensitive to NEM, while only

40% of the specific binding to the 5-HTir in the rabbit CN were sensitive to the reagent. These findings may suggest that differ ent G proteins were involved in these two tissues, since Stratford et al. (1988) found that NEM exerted its effects on 5-HT1A, 5-

HTib. 5-HTjjj binding sites by inactivation the G-proteins or G protein binding site associated with the 5-HT receptor subtypes.

These authors found that the NEM-sensitive sulfhydryl group appeard t:o he located distant from the ligand binding sites.

Further, Stratford et al. (1988) found that with the 5-HTjg site, agonist binding was sensitive to NEM, whereas antagonist binding 91

was not. Also, after NEM treatment of membranes, competition of antagonist binding to the 5-HT|B site by 5-HT was no longer sensi tive to Gpp(NH)p. Finally, they could reconstitute high affinity

[3H]5-HT binding to 5-HTi^ and 5-HTid sites and high affinity 5-HT competition of []-ICYP binding to 5-HTjb sites by addition of naive G-proteins.

As previously discussed, evidence from these studies suggests that [3H]5-HT labels a population of binding sites in rabbit caudate nucleus that is different from the 5-HT1A, S-HTjg,

5-HTjq, 5-HTjj), and 5-HT2 sites. Several additional findings suggest that both the bovine 5-HTjo sites and the rabbit 5-HTjp sites are heterogeneous. These findings are: 1) the competition curves for spiperone and spirilene in bovine CN were best fitted by a two-site model; 2) the inhibition curves for ergotamine and ergocryptine in both rabbit and bovine CN were also best fitted by a two-site model; and 3) the inhibition curves for the dihydroergotamine, dihydroergocristine, and dihydroergocryptine on rabbit

CN were best fitted by a two-site model.

In summary, The present study demonstrates that signifi cant differences exist between the non-5-HT1A/non-5-HTiQ high- affinity [^H]5-HT binding sites in rabbit and bovine CN. The differences exist in both the pharmacological profile and the effects of calcium and GTP. Based on the high-affinity to the 5-

HT of this binding site, it would appear to fall within the 5-HTj 92 category, md as such has been designated by us as 5-HT^r, since at present it has only been demonstrated in the rabbit. The 5-

site also appears to be distinct from several other known 5-

HT receptor subtypes based on its lower affinity for compounds with high affinity and /or selectivity for 5-HT1A (8-OH-DPAT, spiperone), 5-HT1B (RU 24969. TFMPP), S-HT^c (mesulergine), and

5-HT2 (ketanserin and spiperone). Whether the 5-HTiR site repre sents a true 5-HT receptor subtype or whether it is merely a high-affinity binding site for [3H]5-HT remains to be determined.

However, the high affinity for 5-HT, the pharmacologic profile, and the GTP sensitivity strongly suggests the receptor possibili- O ty, and there is ample precedent for novel [ H]5-HT binding sites being shown to be receptors, i.e., the 5-HTj^, 5-HTjg, 5-HTjq, and

5-HTjq sites were all initially identified by binding assays and later found to have functional correlates.

Based on the initial findings presented in this work, several facets of the 5-HTjd and 5-HTjr binding sites remain to be explored. First, whether the 5-HT1D and the 5-HT-^r sites are truly heterogeneous or not will depend on the identification of com pounds which can seperate the compounds of the biphasic inhibition curves seen with the ergots and spiperone/spirilene. Correlated to such a screening effort would be the search for compounds which are highly selective for the 5-HT1D and 5-HT1R sites. Second, as previously mentioned, the identification of functional correlates for the 5-HT|r site would be important for determining the recep 93

tor status of the 5-HTjr site. And third, since the calcium mediated effects on GTP sensitivity of the 5-HTjr site appear to be unique, determining the mechanisms for the apparent differences in the calcium-mediated effects on the GTP sensitivity between the

5-HTjr site and the other 5-HTj subtypes would be directed to wards. 94

CONCLUSIONS

1. [3H]5-HT labels a binding site in rabbit CN that is pharmaco

logically different from the 5-HT1A, 5-HTjg, 5-HTlc, and 5-HT2

sites, and similar to but pharmacologically distinct from the

5-HTjd sites identified in bovine CN;

2. The guanine nucleotide effects on agonist affinity for

[3H]5-HT binding in rabbit CN are markedly different than

those for the S-HTjq site in bovine CN;

3. The interactions of GTP and divalent cations, ie., calcium,

in altering [3H]5-HT binding in rabbit CN is different from

those previously described for 5-HTj sites;

4. The sensitivity of the inactivation by NEM in rabbit CN is

different from that seen for 5-HT1D in bovine CN;

5. Both the 5-HT1D and 5-HTjr binding sites may be heterogeneous

binding sites. 95

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