BRAIN RESEARCH

E L S E V I E R Brain Research 729 (t996) 281-284

Short communication

Distribution and characterisation of the [3H](S)-zacopride labelled 5-HT 3 receptor in pig forebrain

Stephan i e F l e t ch e r *, N i c h o l a s M. Ba rn es

Department (~[ Pharmacology, The Medical School, Unicersi O, of Birmingham. Edgbaston, Birmingham B I 5 277", UK

Accepted 21 May 1996

Abstract

The present study demonstrates the presence and distribution of [3H](S)-zacopride labelled 5-HT 3 (5-hydroxytryptamine3) receptors in pig forebrain. The pharmacological characterisation of 5-HT3 receptor recognition sites in homogenates of pig cerebral cortex provides further evidence for inter-species variation in the pharmacology of the 5-HT 3 receptor.

Keyuords: Serotonin 5-HT 3 receptor; (S)-Zacopride; Radioligand binding; Pig brain

Unlike all other known 5-HT receptors, which are predicted to be 7 transmembrane domain macromolecules coupled to G proteins [12], the 5-HT 3 receptor is a ligand- gated ion channel [12]. To date, only one cDNA sequence encoding a 5-HT 3 receptor subunit has been cloned (the 5-HT3-A receptor subunit [15]), along with a splice variant (designated 5-HT3-As; [11]), which are both murine in origin. Species homologs (rat; [13], human; [5,17]) subse- quently detected display a predicted amino acid sequence homology between 82-98% with the murine subunit, which may account for some of the marked inter-species phar- macological and electrophysiological differences in the 5-HT 3 receptor (for reviews see [6,19]). It should be noted, however, that evidence for intra-species differences with respect to the 5-HT 3 receptor is now considerable (e.g. [20]) such that the presence of additional 5-HT 3 receptor subunits remains attractive. The present study investigated the pharmacology and distribution of [3H](S)-zacopride- labelled 5-HT 3 receptors in the pig brain in the search for additional species differences with respect to this in- doleamine receptor. Preliminary reports of the data have been presented to the British Pharmacological Society [16].

Abbreviations: 5-HT, 5-hydroxytryptamine; 5-HT 3 receptor, 5-hy- droxytryptamine~ receptor; 2-Me-5-HT, 2-methyl-5-hydroxytryptamine; Bm~x, maximum binding at equilibrium; PKd, --lOgl0 molar equilibrium dissociation constant; PBG, phenylbiguanide; mCPBG, meta-chlorophen- ylbiguanide.

* Corresponding author. Fax: +44 (121) 414-4509; E-mail: s.fletcher@bham.ac.uk

Pig brain tissue was obtained from a local abattoir within 30 min of death and transported over ice. Tissues were dissected and frozen at -80°C within 2 h of death. To prepare the radioligand binding homogenate, brain tissues were gently thawed and homogenised (Polytron blender, full power; 10 s) in ice-cold Tris/Krebs buffer (raM; Tris, 50.0; NaCI, 118.0; KC1, 4.75; KH2PO 4, 1.2; MgSO 4, 1.2; CaC12, 2.5; NaHCO 3, 25.0: glucose, 11.0; pH 7.4). The homogenate was washed twice by centrifuga- tion (48000 × g, 10 rain, 4°C) and subsequent resuspen- sion of the pellet in Tris/Krebs buffer, and finally resus- pended at a concentration of 500 mg original wet weight ml i. Protein content was assayed by the Bio-Rad Coomassie Brilliant Blue method, using bovine serum albumin as the standard.

For radioligand binding assays, test tubes (in triplicate) contained 50 i, zl of competing drug or vehicle (Tris/Krebs buffer; total binding) and 100 pA of [3H](S)-zacopride (0.2-21.4 nM for saturation studies and between 0.4-1.5 nM for competition and distribution studies). Brain ho- mogenate (50 ~1) was added to initiate binding, which was allowed to proceed at 37°C for 60 rain (sufficient to reach equilibrium) before termination by rapid filtration (Brandel cell-harvester) under vacuum through pre-wet (0.1% v / v polyethyleneimine in Tris/Krebs buffer) Whatman G F / B filters followed by washing with ice-cold Tris/Krebs buffer (wash time 2 X 8 s: wash volume 2 X 5 ml). Bound ra- dioactivity remaining on the filters was assayed by liquid scintillation spectroscopy. Pargyline (10 ~M) was in- cluded for competition studies using 5-HT or 2-Me-5-HT

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282 S. Fletcher, N.M. Barnes/Brain Research 729 (1996) 281 284

(pargyline at this concentration did not interfere with binding; data not shown).

Saturation and competition data were analysed by com- puter assisted iterative curve fitting according to the equa- tion; b = (Bmax[L]n)/([L]" + (K)n), where b = bound ra- dioligand; Bma ~ = maximum binding at equilibrium; for saturation studies K = molar equilibrium dissociation con- stant or for competition studies K--molar concentration of competing compound to reduce the specific binding by 50%; for saturation studies L = free molar concentration of radioligand or for competition studies L = molar con- centration of competing compound; n -- Hill coefficient.

BRL46470 (endo-N-(8-methyl-8-azabicyclo[3.2.1 ]oct-3- yl)2,3-dihydro-3,3 dimethyl-indole-l-carboxamide hydro- chloride; SmithKline Beecham), cocaine (HC1; Research Biochemicals (RBI)), GR67330X ((+)l,2,3,9-tetrahydro- 9-methyl-3-[(5-methyl- 1 H-imidazol-4-yl)methyl]-4H-carbazol-4-c Glaxo), GR 119566X (1,2,3,9-tetrahydro-3-[(5-metbyl- l H- imidazol-4-yl)methyl]-9-(3-aminopropyl)-4 H-carbazol-4-one; Glaxo), granisetron (HC1; SmithKline Beecham), 5-HT (bimaleate; Sigma), MDL 72222 (3-tropanyl-3,5-dichloro- benzoate; RBI), meta-chlorophenylbiguanide (HC1; mCPBG; RBI), 2-methyl-5-HT (maleate; RBI), metoclo- pramide (He1; RBI), ondansetron (hydrochloride dihy- drate; Glaxo), phenylbiguanide (PBG; Aldrich), quipazine (dimaleate; RBI), SDZ 206-830 ((3o~-homotropanyl)-l- methyl-5-fluro-indole-3-carboxylic acid ester; Sandoz), tropisetron (ICS 205-930; (lo~H,3c~,5c~H-tropan-3-yl)l- H-indole-3-carboxylic acid ester; Sandoz) and (S)- zacopride (HC1; Delalande) were dissolved in a minimum quantity of distilled water and diluted with Tris/Krebs buffer, d-Tubocurarine (chloride; 10 mg/ml; Wellcome) was supplied in aqueous solution and diluted with Tris/Krebs buffer. [~H](S)-Zacopride (78 Ci mmol i, Amersham) was supplied in ethanol and diluted in Tris/Krebs buffer. All drugs and reagents were used as received.

In pig cerebral cortex homogenates, [3H](S)-zacopride (0.2-21.4 nM) labelled an apparently homogenous, sat- urable population of specific binding sites with nanomolar a f f i n i t y (Bma x = 465 _+ 47 fmol g-L wet weight, pK d = 8.85 + 0.11, Hill coefficient = 1.06 _+ 0.12, mean + S.E.M., n = 6; non-specific binding defined by on- dansetron, 10 ~M, Fig. 1A). In competition studies, a range of structurally diverse 5-HT 3 receptor ligands com- peted for approximately 70-95% of [3H](S)-zacopride (ap- proximately 1 nM) binding in pig cerebral cortex ho- mogenates with differing affinities (Table 1, Fig. 1B). The binding displayed a pharmacological profile broadly com- parable to the radiolabelled 5-HT 3 receptor recognition site expressed by other species (e.g. murine NG 108-15 cells (e.g. [9]), human brain (e.g. [1,9]), rat entorhinal cortex (e.g. [2,14]), and mouse cortex (e.g. [8,18])). However, some pharmacological differences were apparent. For ex- ample, d-tubocurarine displayed approximately 10-60 fold lower affinity for the 5-HT 3 receptor expressed in pig

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Fig. 1. (A) Representative saturation data for [3H](S)-zacopride (0.2-21.4 nM) binding to homogenates of pig cerebral cortex. Values represent the specific binding calculated from triplicate determinations of total and non-specific binding (defined by the presence of ondansetron, 10 I~M). Inset: subsequently derived Scatchard plot (B, specifically bound radioli- gand (fmol g i wet weight protein); B/F , specifically bound radioli- gand/free concentration of radioligand (fmol g- i wet weight nM- i)). (B) Competition for [3H](S)-zacopride binding (approximately I nM) in pig cerebral cortex homogenates by a variety of compounds; ~ cocaine, C) granisetron, z~ 5-HT, • mCPBG, [] ondansetron, • d-tubocurarine. Data represents the mean _+ S.E.M., n - 3 - 6 . For pK i values and Hill coefficients see Table I.

cerebral cortex compared to that expressed in NG108-15 cells [9], rat entorhinal cortex [2] and mouse entorhinal cortex [18], but approximately 40 fold higher affinity compared to the 5-HT 3 receptor expressed in human puta- men [9]. Furthermore, the structural analogue of the high affinity compound GR67330X, GR119566X [7], exhibited an affinity for the 5-HT 3 receptor expressed in pig cerebral cortex almost 100 fold lower than that for the 5-HT3 receptor expressed in NG 108-15 cells [7] or rat brain (pK i = 10.2; David Bull, personal communication). More

S. Fletcher, N.M. Barnes/Brain Research 729 (19961 281 284 283

Table 1 The affinities and Hill coefficients of various competing compounds for [3H](S)-zacopride (approximately 1 nM) binding to homogenates of pig cerebral cortex. Data represent the mean ± S.E.M., n = 3-9

Compound p Ki Hill number

GR67330X 9.50 _+ 0.19 0.69 _+ 0.14 Granisetron 9.24 _+ 0.03 1.27 +_ 0.07 SDZ 206-830 9.13 + 0.02 1.30 ± 0.08 (S)-Zacopride 9.08 + 0.06 0.77 ± 0.13 Quipazine 9.02 +_ 0.10 0.67 _+ 0.06 GR119566X 8.21 ±0.06 0.91 ±0.09 mCPBG 8.14 ± 0.07 0.93 ± 0.08 BRL 46470 7.72 ± 0.03 1.08 ± 0.04 Ondansetron 7.71 ± 0.16 1.07 ± 0.13 Tropisetron 7.44 + 0.05 0.90 ± 0.03 MDL 72222 6.33 ± 0.01 0.67 ± 0.08 5-HT 6.28 ± 0.02 0.92 ± 0.05 PBG 6.17 +_ 0.02 1.00 + 0.03 d-Tubocurarine 5.97 _+ 0.05 1.13 ± 0.21 2-Me-5-HT 5.95 ± (/.04 0.87 4 0.05 Metoclopramidc 5.89 + 0.01 1.18 ± O. 14 Cocaine 4.79 ± 0.02 1.16 ± 0.26

minor pharmacological differences were also evident with some other compounds (e.g. tropisetron and MDL 72222

exhibited affinities for the porcine 5-HT 3 receptor which were approximately 40 fold lower than those for the 5-HT 3 receptor expressed in rat entorhinal cortex [2]). It is there-

fore apparent that the pig expresses a further pharmaco- logical variant of the 5-HT 3 receptor.

In the present study, all the investigated compounds competed for [3H](S)-zacopride binding in a manner that

produced Hill coefficients that were not significantly dif- ferent from unity, except for the partial 5-HT 3 receptor agonist quipazine [10]. (It should be noted, however, that GR67330X and MDL7222 exhibited similarly low Hill coefficients, although these failed to reach statistical signif- icance). This is consistent with earlier studies using this

radioligand to identify 5-HT 3 recognition sites originating from other species (e.g. [2-4]), although contrasts data generated with some structurally different 5-HT 3 receptor radioligands (e.g. [3,4]). The reason for the low Hill coeffi- cient generated by quipazine remains unclear, and warrants

further investigation. In addition to inter-species pharmacological differences,

species variations in the relative distribution of central 5-HT 3 receptors are also apparent (for review see [6]). The distribution of specific [3H](S)-zacopride binding in pig

brain provides further evidence of inter-species differences with respect to the 5-HT 3 receptor, with highest levels of [3H](S)-zacopride binding detected in the pig cerebral cortex (from 384 _+ 24 to 298 _+ 22 fmol g - ~ wet weight in the parietal and entorhinal cortices, respectively, mean + S.E.M., n = 3) and amygdala (268_+ 19 fmol g - l wet weight, mean +__ S.E.M., n = 3) with lower levels being detected in other brain regions, including the hippocampus (169_+ 11 fmol g ~), nucleus accumbens (155 + 9 fmol

g-~ ), caudate nucleus (144 +_ 1 1 fmol g-~) , putamen (126 + 1 2 fmol g i), thalamus (65_+ 12 fmol g - l ) and cere-

bellum (37 _+ 9; fmol g-L wet weight, mean + S.E.M.,

n = 3 1 . In conclusion, the present studies have identified spe-

cific [3H](S)-zacopride binding in pig forebrain. The radio-

labelled site was differentially distributed throughout the pig forebrain with highest levels in the cerebral cortex. Within the cerebral cortex the [3H](S)-zacopride binding

site displayed a pharmacological profile broadly compara- ble with the 5-HT~ receptor expressed in other species.

Acknowledgements

We thank Drs. T.P. Blackburn (SmithKline Beecham), D. Bull (Glaxo), G.J. Kilpatrick (Glaxo), J.-C. Levy (Dela-

lande) and W.S. Phillips (Sandoz) for the generous gift of drugs. We also thank Dr. D. Bull tbr sharing unpublished

data. We are also grateful to Mr. P.J. McWilliams tot technical assistance. S. Fletcher is recipient of an A.J. Clark Studentship from the British Pharmacological Soci-

ety.

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