Naunyn-Schmiedeberg's Arch Pharmacol (1995) 352:451-454 ~© Springer-Verlag 1995

K. Fink - J. Zentner • M. G6thert

Subclassification of presynaptic 5-HT autoreceptors in the human cerebral cortex as 5-HTID p receptors

Received: 24 May 1995/Accepted: 15 June 1995

Abstract Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) re- ceptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes prein- cubated with [3H~5-HT were superfused and tritium overflow was stimulated by high K +. The K +-evoked tritium overflow, which was Ca 2 +-dependent but tetro- dotoxin-resistant, was concentration-dependently inhi- bited by the nonselective 5-HT~D~/1D~ receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentra- tion which should block the 5-HT~D~ but not the 5- HT1D~ receptor failed to antagonize the inhibitory ef- fect of 5-carboxamidotryptamine. In contrast, the non- selective 5-HTm~/~D~ receptor antagonist, me- thiothepin, at a concentration which should block both the 5-HT~D~ and the 5-HT~D~ receptor abolished the effect of 5-carb0xamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has pre- viously been classified as 5-HT~D, belongs to the 5-HT1D~ subtype.

Key words Presynaptic 5-HT autoreceptors • Human cerebral cortex • 5-Hydroxytryptamine release • 5-HT~D~ receptor • Ketanserin

Introduction

Serotonin (5-hydroxytryptamine; 5-HT) release in the central nervous system is modulated via inhibitory presynaptic 5-HT receptors (Moret 1985; Middlemiss 1988; Starke et al. 1989). Such 5-HT autoreceptors have also been identified in the human cerebral cortex. In

K. Fink • M. G6thert (I:~) Institut ffir Pharmakologie und Toxikologie der Universitfit Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany J. Zentner Neurochirurgische Klinik der Universit~it Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany

a first investigation in human cerebral cortical slices designed to provide basic evidence for their existence (Schlicker et al. 1985), it was shown that 5-HT inhibits the electrically evoked 5-HT release in a manner sus- ceptible to blockade by methiothepin, a non-selective 5-HT1/2 receptor antagonist. Methiothepin, given alone, facilitated 5-HT release, probably by preventing 5-HT from activating the inhibitory receptors. In fur- ther studies in human cerebral cortical slices (Galzin et al. 1992) and synaptosomes (Maura et al. 1993), these receptors were classified as 5-HT1E) and their presynap- tic location was proved.

According to molecular biological studies, human 5-HT1D receptors are heterogeneous. Two subtypes, termed 5-HT1D~ and 5-HTmp can be distinguished (for review, see Hoyer et al. 1994). They can be dis- criminated by ketanserin, which has a 71-fold higher affinity for cloned human 5-HT~D~ than 5-HT~D ~ recep- tors (Weinshank et al. 1991), and which is assumed to act as an antagonist not only at 5-HT2A, but also at 5-HT~D~ receptors (Kaumann et al. 1994). In contrast to ketanserin, the affinities of other 5-HT receptor agonists (such as 5-carboxamidotryptamine) and an- tagonists (such as methiothepin) for both 5-HT~D re- ceptor subtypes are very similar (Weinshank et al. 1991).

The aim of the present study was to subclassify the presynaptic 5-HT1D autoreeeptor by means of ketan- serin at a concentration which should selectively block the 5-HTID~ receptor and methiothepin at a concentra- tion which should block both the 5-HT1D~ and the 5-HT~I)~ receptor. Some of the results have been re- ported at the Third I U P H A R Satellite Meeting on Serotonin (Chicago, IL, USA, t994; G6thert et al. 1995).

Methods

Cerebral cortical tissue (temporo-basolateral region) was obtained from 16 male and 11 female patients (age 32 + 2.7 years). They

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suffered from epilepsy resistant to antiepileptic drugs and, hence, underwent neurosurgical treatment. The tissue was plunged into cold (4 ° C) Krebs-Henseleit solution immediately after resection. The solution was composed as follows (mM): NaC1 118, KC1 4.8, NaHCO 3 25, KH2 PO4 1.2, CaC12 1.3, MgSO4 1.2, glucose 11.1, ascorbic acid 0.06, disodium EDTA 0.03, equilibrated with 95% 02 and 5% CO2). The patients, premedicated with flunitrazepam and atropine, had been anaesthetized with thiopentone (for introduc- tion), fentanyl and enflurane or isoflurane (for maintenance). Pan- curonium had been injected to achieve muscular relaxation. The cerebral cortical specimens were part of the tissue which had to be removed for therapeutics reasons. The study was approved by the local ethics committee.

Preparation and supelfusion of synaptosomes. A5%(w/v) homo- genate of cortical tissue was prepared in 0.32 tool/1 sucrose (4 ° C) by means of a Potter-Elvehjem glass homogenizer with a rotating teflon pestle (1000 rpm, 6 strokes/2 min). The homogenate was centrifuged at 1000 x g for 10 min (4 ° C), and 7 ml of the supernatant plus 7 ml of the Krebs-Henseleit solution were incubated for 6 min at 37 ° C. After addition of [3H~ 5-hydroxytryptamine 43 nM, incubation was continued for another 6 min. The labelled suspension was centri- fuged at 600 x g for 10 rain. The resulting pellet was resuspended in 2.5 ml ice-cold Krebs-Henseleit solution (protein content 508 ± 86 l.tg/0.1 mI, determined by the method of Lowry et al. 1951). Aliquots of 100 btl of the synaptosomal suspensions were distributed on Whatman GF/B filters and placed into 24 parallel superfusion chambers. Subsequently, the synaptosomes were superfused with Krebs-Henseleit solution (37°C) containing the 5-HT reuptake in- hibitor fluvoxamine 10 ~tM, for 60 rain at a flow rate of 0.6 ml/min. Stimulation was carried out by elevating the K ÷ concentration from 4.8 to 25 mM (Na + reduced accordingly) for 2 min after 40 min of superfusion. Drugs were added to, and in some experiments Ca 2+ was omitted from, the superfusion fluid from 20 min before stimula- tion until the end of the experiment. At the end of the experiments the radioactivity in the 4-rain superfusate samples and the synapto- somes (extracted with 0.25 M HC1) was determined by liquid scintil- lation counting.

Tritium efftux was calculated as the fraction of the tritium con- tent in the synaptosomes at the beginning of the respective collection period (fractional rate of tritium efflux). To quantify effects of drugs on basal efftux, the fractional rate was determined in the sample collected immediately before stimulation. Stimulation-evoked tritium overflow was calculated by subtraction of the estimated basal etttux from total efflux during the stimulation period and the following 10 min and was expressed as percentage of the tritium content in the synaptosomes at the onset of stimulation (basal efflux was assumed to decline linearly from the sample before to that 12-16 rain after onset of stimulation). Means ± SEM of n experi- ments (each in quadruplicate) are given. For comparison of means an unpaired t-test was applied.

Drugs used. 5-[1,2-3H(N)]-Hydroxytryptamine (specific activity 22.8 or 26.7 Ci/mmol; NEN, Dreieich, Germany); tetrodotoxin (Sigma Chemicals, St. Louis, MO, USA); 5-carboxamidotryptamine maleate (RBI, Natick, MA, USA), ketanserin tartrate (Janssen, Neuss, Germany); methiothepin maleate (Hoffmann-La Roche, Basel, Switzerland); fluvoxamine (Duphar, Weesp, The Netherlands).

Results

In control experiments on superfused cortical synapto- somes preincubated with [-3H]5-hydroxytryptamine, the fractional rate of basal tritium etttux from the 36th to 40th min of superfusion amounted to 0.02 _+ 0.0004/min (corresponding to 0.47 _+ 0.07 nCi /min; n = 6). Omission of Ca 2 ÷ or addition of 300 nM

tetrodotoxin, 110 nM methiothepin or 1 1000 nM 5- carboxamidotryptamine in the absence or presence of 110 nM methiothepin did not affect basal tritium eIttux (results not shown). Ketanserin at 320 nM increased basal efflux by 23.2 +_ 6.1% (n = 14; p < 0.05) and a similar increase was observed when 1-1000 nM 5- carboxamidotryptamine was investigated in the pres- ence of 320 nM ketanserin (n = 6-14; no significant difference from 320 nM ketanserin alone). In two pre- liminary experiments with 1000 nM ketanserin, basal tritium efflux was increased by 44 and 56%, which excluded reliable evaluation of K+-evoked tritium overflow.

Omission of Ca 2 ÷ from the superfusion fluid abol- ished the K ÷ (25 mM)-evoked tritium overflow, where- as 300 nM tetrodotoxin was ineffective (Fig. 1, upper panel). Ketanserin 320 nM and methiothepin 110 nM also did not affect the evoked overflow (Fig. 1, upper panel). 5-Carboxamidotryptamine (10-1000 nM) con- centration-dependently reduced the evoked overflow; this effect was not changed by 320 nM ketanserin but abolished by 110 nM methiothepin (Fig. 1, lower panel).

Discussion

Tritium overflow evoked by high K ÷ from human cortical synaptosomes preincubated with a low [3H]5-HT concentration may be assumed to reflect depolarization-evoked exocytotic release of tritiated and unlabelled 5-HT from the serotoninergic nerve terminals (Raiteri et al. 1990). In agreement with this assumption and previous results in synaptosomes (for review see Starke et al. 1989), release was com- pletely dependent on the availability of extracellular Ca 2+ ions, but independent of propagation of action potentials.

The ability of low concentrations of the 5-HT1B/lO receptor agonist, 5-carboxamidotryptamine, to inhibit 5-HT release confirms previous results obtained with this drug in human cortical slices (Galzin et al. 1992) and is compatible with the operation of a presynaptic 5-HT1D autoreceptor (Maura et al. 1993). Ketanserin at a concentration of 320 nM, which is 4.4 times higher than its Ki value at cloned human 5-HTm~ receptors in cells transfected with this receptor gene, but 16.2 times lower than its Ki value at cloned 5-HTID/~ receptors (see Table 1), did not affect the concentration-response curve for 5-carboxamidotryptamine. In contrast, the effect of 100 and 1000 nM 5-carboxamidotryptamine was abolished by the mixed 5-HTlm/lOe receptor an- tagonist methiothepin at a 4.4 times higher concentra- tion than its Ki value at cloned 5-HT1De receptors (Table 1). According to this pattern of effects of the two antagonists, it may be concluded that the presynaptic 5-HT autoreceptor in the human cerebral cortex be- longs to the 5-HTme subtype.

100"

O "c

-o 50-

O

+ v

Contr Ca 2 + TTX Ket Met -free 0.3/~M 0.32/IM O, 11/~M

100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 I I0 I00 I000 5-Carboxamidotryptamine (nM)

Fig. l Effects of 5-HT receptor ligands, tetrodotoxin and omission of Ca 2+ ions from the superfusion fluid on the K+-evoked tritium overflow from superfused human cortical synaptosomes prein- cubated with [3H]-5-hydroxytryptamine. The synaptosomes were superfused with Krebs-Henseleit solution containing 10 ~tM ftu- voxamine throughout. For stimulation, the K + concentration was increased to 25 mM for 2 min after 40 min of superfusion. Upper panel, Effect of omission of Ca 2 + from, or presence of tetrodotoxin (TTX), ketanserin (Ket) or methiothepin (Met) in, the superfusion fluid from 20 min before stimulation onward. Contr: drug-free con- trols. Means + SEM of 5-6 experiments. *P < 0.001 (compared to the controls). Lower panel. Effect of 5-carboxamidotryptamine (5- CT) in the absence of 5-HT receptor antagonists ([~) and interaction with 320 nM ketanserin (I) or 110 nM methiothepin (N). The drugs were present from 20 min before stimulation onward. Controls: tritium overflow in experiments without 5-CT either in the absence or presence of one of the antagonists. Means + SEM of 6-14 experi- ments. *p < 0.05 (compared to the corresponding controls without 5-CT). +p < 0.05 (compared to the effect of the same 5-CT concen- tration in the absence of an antagonist)

In a previous s tudy on h u m a n cortical synapto- somes, a combina t ion of 1 p M ketanserin and 0.3 pM 5-HT inhibited the K ÷ - e v o k e d [ 3 H ] 5 - H T release to the same extent as 0.3 gM 5-HT alone (Maura et al. 1993). However , the interact ion between ketanserin and 5 -HT deserves further investigation for two rea- sons. 1. N o informat ion is given abou t the effect of drugs, in par t icular of 1 ~tM ketanserin, on basal t r i t ium efflux. In the two prel iminary experiments of the present investigation with this ketanserin concen-

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Table 1 Affinity estimates (Kt) of ketanserin and methiothepin for cloned human 5-HT1D receptor subtypes in cell membrane prepara- tions from LM (tk-) cells stably transfected with the indicated clones (from Weinshank et al. 1991) and concentrations investigated in human cortical synaptosomes (antagonism of 5-carboxamidotryp- tamine; present study)

Antagonist Ki (nM) Concentration investigated (nm)

5-HT1D~ 5-HT1D~

Ketanserin 73 5198 320 (4.4 x Km~ ) Methiothepin 11 25 110 (4.4 x Kib~)

tration, basal tr i t ium efftux was increased by abou t 50%. Such a s t rong increase in basal tr i t ium efflux is hardly compat ib le with a meaningful evaluat ion of evoked [ a H ] 5 - H T release. 2. The effect of 1 pM ketan- serin alone was not determined, which is a par t icular d rawback in view of the suggestion that at this concen- t ra t ion the drug m a y exhibit inverse agonistic p roper ty at the h u m a n 5-HTlo~ receptor (Thomas et al. 1995). In this context, it should be ment ioned that meth io thepin can also act as an inverse agonist at the human 5- HTID~ receptor (Thomas et al. 1995). However , at the concentra t ion used in the present study, neither ketan- serin nor meth io thepin inhibited the evoked 5-HT re- lease (see Fig. 1, upper panel), thus excluding the inter- ference of inverse agonism.

Taken together, the present investigation provides first direct evidence that the presynapt ic 5 -HT auto- receptor in the h u m a n cerebral cortex belongs to the 5-HTlo~ subtype. This conclusion conforms to the sug- gestion that the h u m a n 5-HTzo~ receptor and the rat 5-HT~B receptor (which acts as presynapt ic 5 -HT autoreceptor in the rat brain cortex; Engel et al. 1986) are species homologues (Hart ig et al. 1992), a single amino acid difference being responsible for the phar- macological difference between bo th receptors (Oksen- berg et al. 1992).

Acknowledgement We thank Mrs. H. Burisch and Mrs. I. Konrad for their skilled technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 400) and the European Community (Biotechnology program).

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