5-HT1B receptors modulate release of [3H]dopamine from rat striatal synaptosomes

The effect of the selective r5-HT_1B agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo [3,2-b] pyril-5-one (CP93,129) on the K⁺-evoked overflow of [³H]dopamine was studied in rat striatal synaptosomes loaded with [³H]dopamine. The aim of the study was to investigate the participation of 5-HT_1B receptors in the serotonergic modulation of striatal dopaminergic transmission. The Ca²⁺-dependent, tetrodotoxin-resistant K⁺-evoked overflow of [³H]dopamine was inhibited by CP93,129 (0.01–100 μM) in a concentration-dependent manner (IC₅₀=1.8 μM; maximal inhibition by 35.5% of control). [±]8-OH-DPAT, a 5-HT_1A receptor agonist, [+/–]DOI, a 5-HT₂ receptor agonist, and 2-methyl-5-hydroxytryptamine, a 5-HT₃ receptor agonist, at concentrations ranging from 0.01 μM to 100 μM did not show any significant effect. Neither ketanserin (1 μM and 5 μM), a selective 5-HT₂/5-HT_1D receptor antagonist, nor ondansetron (1 μM), a 5-HT₃ receptor antagonist, changed the inhibitory effect of CP93,129. SB224289, GR55562, GR127935, isamoltane and metergoline, selective and non-selective 5-HT_1B receptor antagonists, in contrast, used at a concentration of 1 μM, antagonized the inhibitory effect of CP93,129 (3 μM and 10 μM). SB224289, a selective 5-HT_1B receptor antagonist, inhibited the effect of CP93,129 in a concentration-dependent manner; the calculated K _i value was 1.8 nM. Our results indicate that in rat striatal axon terminals the K⁺-evoked release of dopamine is regulated by the presynaptic 5-HT_1B heteroreceptors. Received: 7 September 1998 / Accepted: 2 November 1998     

Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: Definition of its binding profile using agonists, antagonists and inverse agonists

The novel benzodioxopiperazine, 5-HT_1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT_1A receptor (K_i = 0.6 nM versus [³H]-8-hydroxy-dipropylamino-tetralin, [³H]-8-OH-DPAT): its affinity at h5-HT_1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22° C, [³H]-S 15535 associated and dissociated from h5-HT_1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a K_d estimate of 3.6 nM. In saturation binding experiments, [³H]-S 15535 displayed a B_max value for h5-HT_1A receptors (1630 fmol/mg), higher than that obtained with the agonist [³H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 μM) reduced the binding of [³H]-S 15535 by only 25% compared with 79% for [³H]-8-OH-DPAT at h5-HT_1A receptors. [³H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (B_max = 820 fmol/mg versus 647 fmol/mg for [³H]-8-OH-DPAT). For both h5-HT_1A and rat 5-HT_1A receptors, the K_i values for competition binding of 15 serotonergic ligands with [³H]-S 15535 was highly correlated with that of [³H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [³H]-S 15535 but not with [³H]-8-OH-DPAT at h5-HT_1A receptors. Similarly, the ‘antagonists’, spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [³H]-S 15535 but not [³H]-8-OH-DPAT. When [³H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 μM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [³H]-S 15535 is a highly selective 5-HT_1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT_1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [³H]-agonists and [³H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [³H]-S 15535 being modulated in an opposite manner to those of agonists. Received: 12 September 1997 / Accepted: 1 December 1997     

Different muscarine receptors mediate the prejunctional inhibition of [3H]-noradrenaline release in rat or guinea-pig iris and the contraction of the rabbit iris sphincter muscle

Summary To investigate the muscarine receptor type mediating inhibition of [³H]-noradrenaline release from the isolated rat and guinea-pig iris we have determined the potency of antimuscarinic drugs to antagonize the methacholine-induced inhibition of [³H]-noradrenaline overflow evoked by field stimulation (3 Hz, 2 min). The prejunctional apparent affinities were compared with those obtained for postjunctional muscarine receptors mediating the methacholine-induced contraction of the isolated rabbit iris sphincter muscle.Prejunctional apparent affinity constants of pirenzepine (6.67), himbacine (8.51), methoctramine (7.92), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.00), hexahydro-difenidol enantiomers (6.92, (R); 5.77, (S)) in the rat iris and methoctramine (7.58) in the guinea-pig iris indicate the presence of M₂ receptors. Although the post-junctional affinity constants in the rabbit iris sphincter of methoctramine (5.93), gallamine (3.92), and 4-DAMP (9.07) confirm our previous suggestions of the presence of M₃-like receptors, the results obtained with the hexahydro-difenidol enantiomers do not agree with that concept. The post-junctional affinity constants of the hexahydro-difenidol enantiomers were not different from the prejunctional values (6.86, (R); 5.55, (S)), indicating a similar and low degree of stereoselectivity for these stereoisomers at both receptor sites (14 and 17, (R)/(S)-ratios, respectively). Hence, the postjunctional muscarine receptor in the rabbit iris sphincter fails to exhibit the high degree of stereo selectivity observed for hexahydro-difenidol enantiomers at M₃ receptors on other smooth muscles.This study was supported by the Deusche Forschungsgemeinschaft (Fu 163/2) Send offprint requests to H. Fuder at the above address     

Purinoceptor-mediated modulation by endogenous and exogenous agonists of stimulation-evoked [3H]noradrenaline release on rat iris

Summary To investigate whether endogenous purinoceptor agonists affect the sympathetic neurotransmission in the rat isolated iris, and to classify the purinoceptors modulating exocytotic [³H]-noradrenaline release, we have determined the effect of adenosine receptor antagonists on, and the relative potency of selected agonists in modulating, the field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow. In addition, the apparent affinity constants of 8-phenyltheophylline (8-PT) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in antagonizing the prejunctional effects of purinoceptor agonists were estimated.The relatively A₁-selective DPCPX 10 and 100 nmol/l increased the evoked [³H]-noradrenaline overflow by about 25%–35%a indicating a minor inhibition of evoked release by endogenous purinoceptor agonists probably via an A₁ adenosine receptor. Whereas the A₁/A₂-antagonist 8-PT failed to increase the evoked [³H]-noradrenaline overflow in the absence of exogenous agonists (without or with dipyridamole 1 pmol/l present), the relatively A₂-selective antagonist CP-66,713 (4-amino-8-chloro -1-phenyl(1,2,4)triazolo(4,3-a)quinoxaline) 100 nmol/l decreased it by 20%–30% in the absence and continuous presence of DPCPX. This may be compatible with a minor A₂-mediated facilitation by an endogenous purinoceptor agonist.All exogenous agonists tested (except UTP 100 mol/l) inhibited the evoked [³H]-noradrenaline overflow. The relative order of agonist potency (IC4o, concentration in mol/l for inhibition of evoked release by 40%) was CPA (N⁶-(cyclopentyl)adenosine, 0.004) > R-PIA (R(–)N6-(2phenylisopropyl)adenosine, 0.066) = CHA (N⁶-(cyclohexyl)adenosine, 0.082) > NECA (N⁵-(ethyl-carboxamido)adenosine 0.44) > ADO (adenosine, 4.1). ATP was n early equipotent with ADO. Maximum inhibition was 70%–80% and similar for all agonists. Adenosine deaminase 1 u/ml failed to affect the ATP-induced, but abolished the adenosine-induced prejunctional inhibition. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine (NBTG) failed to enhance the potency of ADO and ATP. The A₁-selective antagonist DPCPX 10 nmol/l did not reduce the ATP potency indicating an effect of ATP per se not mediated via an A₁ purinoceptor.Prejunctional affinity constants of 8-PT were 6.07 when tested against adenosine (in the presence of dipyridamole), and 6.60 against CHA. The apparent -log K_B of DPCPX tested against CPA was 9.71. The high DPCPX affinity is compatible with an A₁ adenosine receptor mediating inhibition of sympathetic neurotransmission in rat iris. This receptor may not be the only prejunctional purinoceptor on rat iris sympathetic nerves. The receptor by which ATP acts prejunctionally in this tissue remains to be determined.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/2 and 163/3) Send offprint requests to H. Fuder at the above address