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AGONIST AND ANTAGONIST EFFECTS OF 5-HT1A RECEPTOR LIGANDS ON EXCITATORY SYNAPTIC TRANSMISSION IN THE RAT HIPPOCAMPUS IN VIVO D. Manahan-Vaughan*, M.J. Rowan & R. Anwyl, Department of Pharmacology and Therapeutics and Department of Physiology, Trinity College, Dublin 2, Ireland. The relative agonist and antagonist properties of a series of 5-HT receptor ligands administered systemically was assessed electrophysiologically in the hippocampu Aof alert rats. The ability to reduce excitatory synaptic transmission was used as a measure of 5-HT1A receptor mediated responsiveness (O'Connor et al, 1990). A pair of stimulating and recording wire electrodes were implanted in the stratum radiatum of the CA1 region of the dorsal hippocampus of pentobarbitone (40 mg/kg, i.p.) anaesthetized male Wistar rats (200-250 g). Animals were allowed at least 1 week to recover before electrically evoked population excitatory post-synaptic potentials (EPSP) were recorded whilst lightly restrained in a hammock. Changes in tie amplitude of the EPSP (60% maximum) were recorded after i.p. injection of the drugs. 8-OH-DPAT (8-hydrox-2-(di-n-propylamino)te+ralin, 25-75 pg/kg), gepirone (0.5-10 mg/kg) and its metabolite 1-(2-pyrimidinyl)-piperazine (: P, 0.25-1 mg/kg) produced dose-dependent transient reductions in the amplitude of the EPSP with maximum decreases of 73 ± 4% (n = 4), 38 ± 3% (n = 4) and 32 ± 2% (n = 6) respectively. Whereas MDL 73005EF (2 mg/kg, Moser et al, 1990) had no effect on its own it significantly blocked the reduction of the EPSP amplitude produced by either 50 pg/kg 8-OH-DPAT (from 57 ± 6% to 15 ± 2%, n = 4, P < 0.01, mean ± s.e. mean % decrease, Student's t test), 3 mg/kg gepirone (from 27 ± 2% to 5 ± 1% decrease, n = 4, P < 0.01) and 1-PP (from 28 ± 0.1% to 9 ± 2% decrease, n = 4, P < 0.01). Pretreatment with gepirone (3 mg/kg) or 1-PP (1 mg/kg) prevented a further significant reduction in the EPSP amplitude by 50 pg/kg 8-OH-DPAT (26 ± 3% and 20 ± 3% decrease for gepirone + water and gepirone + 8-OH-DPAT respectively; 29 ± 1% and 27 ± 2% decrease for 1-PP + water and 1-PP+8-OH-DPAT respectively, n = 4 per group). These findings suggest that with regard to 5-HT receptor modulation of excitatory synaptic transmission in the hippocampus of the alert rat 8-OH-DPAT b^aves as an agonist, gepirone and 1-PP as partial agonists and MDL 73005EF as an antagonist when given systemically. Moser, P.C., Tricklebank, M.D., Middlemiss, D.N., Mir, A.K., Hibert, M.F. & Fozard, J.R. (1990) Br. J. Pharmacol. 99, 343-349. O'Connor, J.J., Rowan, M.J. & Anwyl, R. (1990) Br J. Pharmacol. 101, 171-177.

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(-)-PENBUTOLOL ANTAGONIZES 8-OH-DPAT-INDUCED INHIBITION OF RAT 5-HYDROXYTRYPTAMINERGIC DORSAL RAPHE NEURONS

C.P. VanderMaelen & J.P. Braselton, CNS Research, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT. 06492, U.S.A. (Introduced by M.J. Rowan) A number of compounds, such as BMY 7378, NAN-190, and RK-153, which display antagonist actions in "postsynaptic" models of serotonin type 1A (5-HT1A) receptor function, appear to act predominantly in an agonist fashion at "presynaptic" 5-HT1A receptors located on cell bodies and dendrites of serotonergic dorsal raphe (DR) neurons (Hjorth & Sharp, 1990; Sharp et al., 1990; VanderMaelen & Braselton, 1991). The 0-adrenoceptor and 5-HT1A ligand (-)-penbutolol [(-)-P] has displayed antagonist actions in biochemical and behavioral studies of 5-HT1A function. A recent microdialysis study by Hjorth et al., (1991) suggests that (-)-P does not exert 5-HT1A agonist actions on serotonergic DR neurons, and can block the inhibitory effects of 8-OH-DPAT. The present study examined this question directly, using extracellular single unit recording and microiontophoretic electrophysiological techniques in chloral hydrate anesthetized adult male Sprague-Dawley rats. Intravenous doses of (-)-P, ranging from 0.001 to 2.0 mg/kg produced weak and variable inhibitory effects, with most cells partially inhibited, and a few completely inhibited. Overall, impulse flow was preserved, consistent with the actions of a very weak 5-HT1A partial agonist. Surprisingly, pretreatment of rats for 10 min with 0.5 mg/kg, i.v. of (-)-P had absolutely no antagonist-like effect on the dose-response curve for inhibition of DR neuronal firing induced by 8-OH-DPAT. However, when a protocol similar to that used by Hjorth et al. (1991) was utilized, with (-)-P administered s.c. 60 min or more before 8-OH-DPAT, an 11-fold shift to the right of the 8-OH-DPAT doseresponse curve was observed (ED5o values = 2.13 and 24.8 g±g/kg, i.v.). This high dose of (-)-P by itself produced partial inhibition of firing for most cells tested, but in general, impulse flow was still preserved. Microiontophoretic administration of 8-OH-DPAT produced inhibition of firing of serotonergic DR neurons. This response showed little net change over time following s.c. saline injection. However, the inhibitory response to microiontophoretic 8-OH-DPAT was reduced an average of 85.5% by (-)-P when the same protocol described above was used (60 min or more after 8.0 mg/kg, s.c. (-)-P). These results are consistent with the notion that (-)-P is a very weak 5-HT1 A partial agonist that can effectively antagonize "presynaptic" 5-HT1 A receptors located on serotonergic DR neurons, without shutting down impulse flow in these cells. Why the lower i.v. dose with the shorter pre-treatment time failed to produce any detectable antagonism remains to be explored. (-)-Penbutolol was graciously provided by J. Chamberlain and S. Maayani.

Hjorth, S. & Sharp, T. (1990) Life Sci. 46, 955-963. Hjorth,S., Sharp, T. & Grahame-Smith, D. (1991) Soc. Neurosci. Abst. 17, 91. Sharp, T., Backus, L.I., Hjorth, S., Bramwell, S.R. & Grahame-Smith, D. (1990) Eur. I. Pharmacol. 176, 331-340. VanderMaelen, C.P. & Braselton, J.P. (1991) Soc. Neurosci. Abst. 17, 93.

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THE DIFFERENTIAL PHARMACOLOGY OF THE (-) AND (+) STEREOISOMERS OF WAY 100339 AT 5-HT1A AND ADRENOCEPTORS IN THE RAT

a2-

R.A. Spokes, H.L. Mansell, E.A. Forster, J.E. Hartley, Y. Reilly, N. Bissue, V.C. Middlefell, A. Fletcher & I.A. Cliffe. Department of Biomedical Research,Wyeth Research (UK) Limited, Huntercombe Lane South, Taplow, Maidenhead, Berkshire. SL6 OPH. WAY 100339 (7-(1-azetidinyl)-5,6,7,8-tetrahydroquinoline) was synthesised as part of a programme designed to identify 5-HTIA receptor ligands. Here we report the pharmacological profile of WAY 100339 and its stereoisomers, WAY 100507 (-) and WAY 100508 (+). Standard radioligand binding assays on WAY 100339, WAY 100508 and WAY 100507 demonstrated affinities at 5-HT1A receptors with IC50 s of 136, 60 and 2994 nM and at a2 adrenoceptors of 219, 25 and 458 nM respectively. WAY 100339 produced a typical 5-HT1A behavioural syndrome with an ED50 (95% confidence limits) = 0.77 (0.53-1.2) mg/kg i.v, similar to that produced by the selective 5-HT1A receptor agonist flesinoxan (ED50 = 1.5 (1.1-2.0) mg/kg i.v.). WAY 100507 (10 mg/kg, i.v.) did not produce syndrome. WAY 100508 (0.3-10 mg/kg, i.v.) produced sedation and ataxia at all doses. Co-administration of WAY 100508 and WAY 100507 (1.5 mg/kg, i.v.) produced the 5-HT1A syndrome. Also, when WAY 100508 was co-administered with either of the two a2 antagonists, Wy 26392 (Paciorek et al, 1984) or idazoxan (both at 0.5 mg/kg, i.v.), the 5-HT1A syndrome was evoked. The sedative potentials of WAY 100339 (0.3-10mg/kg), WAY 100508 (0.3-3 mg/kg), WAY 100507 (1-100 mg/kg) and the ai2-adrenoceptor agonist clonidine (0.03-1 mg/kg) were assessed using groups of 6 to 8 rats tested 60 min after oral dosing in automated open fields. All the compounds tested produced dose related decreases in locomotor activity. The doses of each compound required to inhibit locomotor activity to 50 % of control levels were 4.3, 0.53, >100 and 0.14 mg/kg p.o. respectively. In pentobarbitone (70 mg/kg, i.p.) anaesthetised rats, cumulative administration of WAY 100339 (0.01-1 mg/kg, i.v.), WAY 100508 (0.003-1 mg/kg, i.v.) and clonidine (0.3-30 Aig/kg, i.v.) produced dose related falls in MABP (mean arterial blood pressure). These responses and the effects of antagonists on them are tabulated below. WAY 100507 alone (0.01-10 mg/kg) did not signifcantly reduce MABP. Control ED20 95% Confidence Antagonist Dose 95% Confidence Dose ratio Agonist New ED20 (ig/kg) (n=4) limits limits (rg/kg) (n) (mfAg) 115.2-117.8 1.0 Wy 26392 331 WAY 100339 42.4 (6) 124-883** 7.8 0.7-4.8 40.4 1.9 Wy 26392 1.0 21.3 Clonidine 20.1-78.7** (4) WAY 100507 10 2.8-61.7 WAY 100508 13.3 (4) 372 127-1084*** 28.0 WAY 100507 10 '0.5-5.9 '1.7 clonidine response abolished >>18 Clonidine (5) ** P3OuM AlC13 significantly reduced The EC50 which carbachol-induced 4 Ca2 +release was assessed,6 the IC50 for Al was approximately . However, maximal carbachol as a stimulus of 45Ca2+ release (1.9 x 10 M) was not altered by lOOpM AlCl +here was no release of 45Ca2+ dropped from 64% (control) to 24% in the5 presence of 100pM AlCl3. ignificant effect of AlC13 (up to 1mM) on InsP3-induced Ca release. AlCl also inhibitedpotent production with an IC50 of approximately 15DM. 100pM dimethyl hydroxypyridin-I-one (CP20), a aluminium chelator (K =31), was able to abort and reverse (after 2hr incubation) the effects of AlCl3 on both Ca'+ release andSInsP3 production. These data suggest that, in permeabilsied cells, the effect of aluminium on the phosphoinositide-mediated reflect signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may interference with receptor-G protein-phospholipase C coupling (e.g. through a competition of A1 with Mg2+ at the GTP-binding site of the G protein) or an interaction with the lipid vicinal 4,5 phosphates.

carbachol-inV+uced

904M.

InsP

J. Med. 310, 1113-1115. Alfey, C.A. (1984) New Engl. Fairburn, A., Oakley, A.E., Carpenter, T.A., Atack, J.R., Blessed, Candy, J.M., Klinowsky, J., Perry, E.K., G. and Edwardson, J.A. (1986) Lancet, i, 354-357. Oct. 29, 1008-1010. Birchall, J.D. and Chappell, J-S. (198Th Lancet,

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THE AFFINITY OF 8-CYCLOPENTYL-1,3-DIPROPYLXANTHINE AT Al ADENOSINE RECEPTORS OF GUINEA-PIG CEREBRAL CORTEX

S.P.H. Alexander, A.R. Curtis, DA Kendall & SJ. Hill, Department of Physiology & Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG 2UH, UNITED KINGDOM. In guinea-pig cerebral cortical slices, adenosine analogues stimulate cAMP accumulation* (through AZb receptors) and selectively potentiate histamine-stimulated phosphoinositide turnover (through an unidentified adenosme receptor, Ax; Alexander et aL, 1989). We have previously observed that the antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) exhibited ten-fold selectivity at these receptors (Kj Avb 131 nM & Ax 12 nM; Alexander et al, 1989). the present report describes an examination of the affinity of DPCPX at A1 adenosine receptors of guinea-pig cerebral cortex. Accumulation of [3H]cAMP in [3H]adenine-pre-labelled guinea-pig cerebral cortical slices was assayed as previously described (Alexander et aL, 1989). Radioligand binding of [3H]DPCPX was carried out over 90 minutes at 20 C in 50 mM Tris buffer, 1 mM EDTA, 0.01 % Triton X-100, pH 7.4 containing 1-2 U/mL adenosine deaminase, defining non-displaceable binding with 1 mM theophylline. Radoligand bound to a 30 000 g particulate preparation from cerebral cortex was collected by rapid vacuum filtration over Whatman GF/B filters. At least three separate preparations were used for affinity determinations. The adenosine analogue N6-cyclopentyladenosine (CPA) inhibited forskolin (30 pM) -stimulated cAMP accumulation in cerebral cortical slices in a concentration-dependent manner (IC50 16 ± 1 nM). The presence of DPCPX shifted the concentration-response curve to CPA to the right in an a arently parallel fashion to allow estimation of the apparent affinity constant of 6 ± 1 nM. Analysis of saturation isotherms of sH]DPCPX binding to cerebral cortical membranes indicated a single binding site with high capacity (B 1560 ± 278 fmol/mg protein) and with high affinity (Kd 4.2 ± 0.4 nM). DPCPX displaced [3H]DPCPX (2-3 M) binding wZtihigh potency (Kj 4.4 ± 1.9 nM) in an apparently monophasic manner. The agonist CPA displaced [3H]DPCPX in a biphasic manner, with calculated Kj values of 7.3 ± 2.5 and 450 ± 87 nM, the latter site constituting 65 ±8 % of [3H]DPCPX binding. We conclude that the affinity of DPCPX for A receptors of guinea-pig cerebral cortex is comparable in both functional and radioligand binding assays. The similarity of affinities for DPCPX at functional Al and Ax receptors, together with the lack of evidence for multiple antagonist radioligand binding sites, raises the Possibility that the two receptors are identical, although it is also likely that two binding sites of such similar affinities would not be distinguished. The similarity of the affinities of CPA in displacing [3H]DPCPX appears to align the high affinity site (7 nM) with the A1 rece tor (16 nM) and the low affinity site (450 7). It is tempting to specu ate therefore that whereas the antagonist nM) with the A receptor (410 nM; Hill & end, DPCPX is unable to distinguish between these receptors, the agonist CPA may be useful to define these receptors in radioligand binding studies. However, the question of the identity of the Ax receptor must await further investigation. The financial support of the Wellcome Trust is gratefully acknowledged. Alexander, SPH; Kendall, DA & Hill, SJ (1989) Br J Pharmacol 98, 1241-1248 Hill, SJ & Kendall DA (1987) Br J Pharmacol 91, 661-669

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CAN Ni2+ BE USED TO DEFINE THE COMPONENT DEPENDENT ON Ca2+ ENTRY IN HISTAMINE-INDUCED INOSITOL PHOSPHATE FORMATION IN BRAIN?

J.A. Arias-Montafio & J.M. Young, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ.

Histamine-induced inositol phosphate (IP) formation in mouse and rat cerebral cortex is strongly dependent on the extracellular concentration of Ca2+ (Alexander et al., 1990). The same is true, but apparently to a lesser extent in guinea-pig cerebral cortex and cerebellum. The analysis of these responses would be aided if it were possible to selectively block Ca2+ entry. The utility of divalent cations such as Ni2+ for this purpose is uncertain, since they are effective inhibitors of [3H]-mepyramine binding to histamine Hrreceptors in homogenates of guinea-pig cerebellum (Treherne et al., 1991). However, it is not certain that non-cell-penetrant ions such as Ni2+ will inhibit histamine binding to H -receptors in intact tissues. The apparent lack of a Ca2+ influx component in histamine- and carbachol-induced IP formation in HeLa cells at extracellular Ca2+ concentrations above 0.3 mM (Arias-Montaflo & Young, (1992) offers the opportunity to test whether Ni2+ inhibits HI-receptor function in intact cells. HeLa cells were cultured and labelled with [3H1-inositol as described previously (Bristow et al., 1991). Incubations with histamine or carbachol in the presence of 30 mM Li+ were for 30 min. Cross-chopped slices of regions of guinea-pig or rat brain were incubated with [3H]-inositol and 10 mM Li+ before addition of 1 mM histamine and further incubation for 60 min. Incubations were terminated by addition of 10% perchloric acid and [3H]-inositol phosphates separated by anion-exchange chromatography. Inhibition of [3H]-mepyramine binding to rat cerebral cortical homogenates was measured as described by Treherne et al. (1991).

Ni2+ inhibited histamine-induced [3H]-IP1 accumulation in slices of rat cerebral cortex and guinea-pig cerebral cortex and cerebellum with IC50s of 0.4 and 1 mM in rat and guinea-pig, respectively. The effect of 1 mM Ni + on the histamine concentrationresponse curve in rat cerebral cortex was to reduce the maximum response, consistent with non-competitive inhibition. Ni2+ also inhibited [3H]-mepyramine binding to homogenates of rat cerebral cortex, the dependence on radioligand concentration being consistent with allosteric inhibition (IC50 with 0.5 nM [3H]-mepyramine 0.5 mM). In HeLa cells Ni2+ (1 mM) had no significant effect on the concentration-response curve for carbachol-stimulated [3H]-IP formation. However, 1 mM Ni2+ caused a parallel displacement to the right of the concentration-response curve for histamine. ½he curve was displaced further by increasing the concentration of Ni2+ to 2 and 3 mM. The inhibition by Ni2+ of histamine-induced [3H]-IPI in HeLa cells, compared with the lack of effect on the response to carbachol, which shows the same pattern of Ca2+-dependence, strongly suggests that Ni2+ exerts an inhibitory action on H l-receptor function by acting at a site accessible from the extracellular medium. This makes it unlikely that Ni2+ can be used to define the Ca2+ entry component in histamine-induced IP formation in brain tissues. Alexander, S.P., Hill, S.J. & Kendall, D.A. (1990) Biochem. Pharmacol. 40, 1793-1799. Arias-Montafto, J.A. & Young, J.M. (1992) Br. J. Pharmacol. 105, 23P. Bristow, D.R., Arias-Montaflo, J.A. & Young, J.M. (1991) Br. J. Pharmacol. 104,677-684. Treheme, J.M., Stem, J.S., Flack, W.J. & Young, J.M. (1991) Br. J. Pharmacol. 104, 1745-175 1.

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ENDOGENOUS LIPOCORTIN-1 MEDIATES DEXAMETHASONE INHIBITION OF INTERLEUKIN-1-INDUCED NEUTROPHIL ACCUMULATION IN VIVO

M. Perretti, J.D. Croxtall and R.J. Flower. Department of Biochemical Pharmacology, The William Harvey Research Institute, The Medical College of Saint Bartholomew Hospital, Charterhouse Square, London EC1M 6BQ. We have recently described lipocortin-1 (LC1) and dexamethasone (Dex) as potent inhibitors of interleukin-1 (IL-i) elicited neutrophil (PMN) accumulation into the mouse air-pouch (Perretti and Flower, 1992). In the present study we have investigated the possibility that endogenous LC1 could mediate this Dex effect. We have optimized the conditions for the appearence of anti-LC1 activity in the blood of mice after treatment with a polyclonal sheep antiserum (50 ,j1 s.c.) by using a specific ELISA (Goulding et al., 1989). A peak of anti-LC1 activity was found at 24h (titre 39,000) with levels remaining high for approximately 150h after which they declined rapidly becoming undetectable by 650h post injection. Air-pouches were formed on the back of male Swiss mice (20-22g) by injection of 2.5 ml of air on day 0 and day 3 (Perretti and Flower, 1992). Antibodies prepared against human LC1 were injected on day 5 (50 ul s.c. for non-immune, pre-immune and specific anti-LC1 sheep sera; 100 ,g s.c. for mouse IgG and monoclonal antibody (mAb) 1A and 1B, Biogen, Cambridge, MA). After 24h, Dex (5 jig) was administered i.v., and 20 ng IL-1,B (I.R.I.S., Siena, Italy) given locally 2h after the steroid. PMN accumulation was evaluated 4h following treatment with the cytokine by staining in Turk's solution the lavage fluids (1). IL-1f3-induced migration (6.30 + 0.56 x106 PMN per mouse, mean + S.E., n=23) was reduced by 52% after Dex treatment (3.03 + 0.33 x106, n= 12, p