Irreversible blockade of central 5-HT1A receptor binding sites by the photoaffinity probe 8-methoxy-3'-NAP-amino-PAT

A new photoaffinity ligand derived from the potent 5-HT agonist, 8-OH-DPAT, has been synthesized. In the dark, this compound, 8-methoxy-2-(N-n-propyl,N-3-(2-nitro-4-azidophenyl)aminopropyl) aminotetralin or 8-methoxy-3'-NAP-amino-PAT, displaced [3H]8-OH-DPAT and [3H]5-HT bound to 5-HT1A and 5-HT1 sites in hippocampal membranes with IC50 values of 6.6 and 18.1 nM respectively. The apparent affinity of 8-methoxy-3'-NAP-amino-PAT for the 5-HT1A binding sites was at least 20 times higher than for the other 5-HT receptor sites (5-HT2 and 5-HT3) or the dopamine-related [3H]spiperone and [3H]7-OH-DPAT binding sites. Under UV irradiation (lambda = 366 nm), 8-methoxy-3'-NAP-amino-PAT produced an irreversible blockade of 5-HT1A sites which could be prevented by prior site occupancy by a saturating concentration (10 microM) of reversible 5-HT ligands such as 5-HT itself, 8-OH-DPAT or LSD. The blockade of 5-HT1A binding sites was concentration-dependent, and two successive irradiations of rat brain membranes in the presence of 30 nM 8-methoxy-3'-NAP-amino-PAT were found to be more efficient that a single exposure to 100 nM of the photosensitive ligand. Thus, a 55-60% irreversible blockade of 5-HT1A binding sites was achieved following 2 cumulative irradiations of hippocampal membranes with 30 nM 8-methoxy-3'-NAP-amino-PAT. Under such conditions, cortical 5-HT2 receptor binding sites as well as striatal 5-HT3 and dopamine-related binding sites remained unaltered.     

Differential selectivities of RU 24969 and 8-OH-DPAT for the purported 5-HT1A and 5-HT1B binding sites. Correlation between 5-HT1A affinity and hypotensive activity

RU 24969 and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) inhibited the specific binding of [³H]5-HT (2 nM) to rat brain membranes with shallow displacement curves. The displacement data were best fitted with a model of two independent, high and low affinity binding sites. Following addition of spiperone (1 μM) as a selective ligand for the putative 5-HT_1A recognition site of [³H]5-HT, the displacement curve of RU 24969 underwent a leftward shift, whereas spiperone induced a shift to the right for the displacement curve of 8-OH-DPAT. In contrast to spiperone, pindolol (1 μM) shifted the displacement curve of RU 24969 to the right. These results suggest that RU 24969 possesses preference for the purported 5-HT_1B subtype of central 5-HT₁ recognition site. The reported significant linear correlation between hypotensive activity following intravenous (i.v.) administration to anesthetized rats and affinity for the central 5-HT₁ binding site could only be maintained by incorporation of the affinity of RU 24969 for its low and 8-OH-DPAT for its high affinity binding site. Based on the proposal that the 5-HT_1A site corresponds to the high affinity site of 8-OH-DPAT and the low affinity site of RU 24969, it is hypothesized that the late depressor phase of 5-HT agonists in rats is mediated by activation of peripheral (vascular) 5-HT receptors which have similarities with the 5-HT_1A subtype of central 5-HT₁ recognition site.     

Modulation of neuronal activity in the hippocampus by 5-hydroxytryptamine and 5-hydroxytryptamine1A selective drugs

The interactions between 5-hydroxytryptamine (5-HT), 8-hydroxy-2-(N,N-dipropylamino)-tetralin (8-OH-DPAT), buspirone, 2-(4-(4-(2-pyrimidinyl)-1-piperazinyl)butyl)-1,2-benzisothiazol-3- (2H)one-1, 1-dioxide-hydrochloride (TVX Q 7821) and ketanserin, and putative 5-HT receptors were analyzed using both radioligand techniques and an in vitro hippocampal slice preparation. The potencies of the drugs were determined at 5-HT1A binding sites labelled by [3H]8-OH-DPAT in hippocampal membranes from the rat. The binding site had similar affinity for 5-HT, 8-OH-DPAT, buspirone and TVX Q 7821, whereas ketanserin was essentially inactive. Physiological effects of these drugs were also examined using an in vitro hippocampal slice preparation. With the exception of ketanserin, application of each drug to the bath modulated the amplitude of the field potential recorded in the pyramidal layer of CA1 evoked by stimulation of Schaffer collaterals. Application of micromolar concentrations of 5-HT produced an initial increase in the population spike followed by a return to near baseline levels within 5 min. By contrast, the amplitude of the population spike was reduced in a dose-dependent manner by micromolar concentrations of 8-OH-DPAT, buspirone and TVX Q 7821, beginning 5 min after application of drug. Ketanserin did not affect the amplitude of the population spike and it did not antagonize the effects of 5-HT, buspirone or TVX Q 7821. Neither buspirone nor 8-OH-DPAT altered the initial increase in population spike induced by 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)     

The guinea-pig ileum preparation as a model for 5-HT1A receptors: anomalous effects with RS-30199-193.

1. Agents that have high and selective affinity for the 5-HT1A site such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and N,N-dipropyl-5-carboxamidotryptamine (DP5CT) inhibited the responses to field stimulation in guinea-pig ileum preparations; the inhibitory effects were antagonized by methiothepin and spiperone, consistent with effects at the 5-HT1A site. 2. The inhibitory effects of DP5CT were pronounced in Tyrode solution containing low Ca2+ (0.9 mM), but were much less apparent in Tyrode solution containing 1.8 or 5.4 mM Ca2+. 3. Responses to DP5CT were abolished by pretreatment with phorbol dibutyrate (3 microM), whereas the responses to UK14304 were only slightly inhibited. 4. Buspirone and ipsapirone (1 microM) inhibited the responses to field stimulation, and the effects were resistant to idazoxan, but inhibited by 8-OH-DPAT or spiperone. 5. RS-30199-193 (5-chloro-2-methyl-1,2,3,4,8,9,10,10a-octahydronaphth-[1,8-cd]- aze pine hydrochloride) an azepine with high affinity for the 5-HT1A site in rat cerebral cortex in binding experiments, augmented contractions, but did not antagonize the responses to DP5CT or to 8-OH-DPAT. 6. The hybrid compound of RS-30199-193 with buspirone, RS-64459-193 (5-chloro-2-[4-(8-azaspiro[4,5]decane-7,9-dione)-but-1-yl]- 1,2,3,4,8,9,10,10a-octahydronaphth[1,8-cd]-3-azepine hydrochloride) maintained high affinity for the 5-HT1A binding site in rat brain and both inhibited the response to field stimulation and antagonized the responses to 8-OH-DPAT and DP5CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

S 14506: novel receptor coupling at 5-HT(1A) receptors

S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

Investigation of mixed D2/5-HT1A activity of N-heteroarylmethyl-N-phenylpiperazines, N-heteroarylethyl-N-phenylpiperazines and N-heteroarylpropyl-N-phenylpiperazines.

Eight novel N-heteroarylalkyl-N-phenylpiperazines have been synthesized, chemically characterized and evaluated for in vitro binding affinity at the dopamine and serotonin receptors. Synaptosomal membranes of fresh bovine caudate nuclei (D1 and D2), the membranes of COS-7 cells (D4.4) and those prepared from fresh bovine hippocampi (5-HT1A) were used as a source of the corresponding receptor subtypes. [3H]SCH 23390 (D1-selective), [3H]spiperone (D2- and D4.4-selective) and [3H]-8-OH-DPAT (5-HT1A-selective) served as radioligands. None of the compounds expressed the affinity for the binding at the D1 subtype receptor. Compounds 7-9 containing a single methylene group serving as a bridge between heteroaryl- and N-phenylpiperazine part of the molecule were inactive [3H]spiperone and [3H]-8-OH-DPAT competitors. Ligands 15-19 (three methylene groups connecting heteroaryl- and N-phenylpiperazine part of the molecule) acted as moderate competitors of [3H]spiperone binding at the D2 receptor subtype, with the exception of 15 (a thione) which expressed a high binding affinity at the D2 receptor subtype. Compounds 15-19 behaved as moderate displacers of 8-OH-[3H]DPAT. Among all eight novel ligands only compound 15 expressed a moderate binding affinity at the D4.4 receptor subtype.     

Molecular pharmacology of 5-HT1D recognition sites: Radioligand binding studies in human,pig and calf brain membranes

1) The binding characteristics of [3H]5-HT (5-hydroxytryptamine, serotonin) were investigated in membrane preparations of several regions from calf, pig and human brain in the presence of 100 nmol/l 8-OH-DPAT (8-hydroxy-2[di-n-dipropylamino]tetralin) and 100 nmol/l mesulergine in order to mask 5-HT1A and 5-HT1C sites. 2) [3H]5-HT bound rapidly, reversibly and stereo-selectively to a population of high affinity recognition sites in membranes from pig caudate, calf caudate and human cortex, caudate and substantia nigra. 3) Saturation experiments carried out with [3H]5-HT in the presence of 100 nmol/l 8-OH-DPAT and 100 nmol/l mesulergine revealed that non-5-HT1A non-5-HT1C sites represented from 50 to more than 90% of the total 5-HT1 sites (determined with [3H]5-HT in the absence of 8-OH-DPAT and mesulergine), depending on the tissue source. 4) The pharmacological profile of these sites was characterized in competition experiments performed with a variety of ligands in membranes of calf, pig and human caudate membranes. Under these conditions, [3H]5-HT labelled a population of "5-HT1-like" sites which display nanomolar affinity for tryptamines (5-carboxamidotryptamine greater than 5-HT greater than or equal to 5-methoxytryptamine greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of subacute treatment with toluene on central monoamine receptors in the rat. Reduced affinity in [3H]5-hydroxytryptamine binding sites and in [3H]spiperone binding sites linked to dopamine receptors

Subacute treatment with toluene leads to a dose-related reduction of affinity in the [3H]spiperone binding sites labeling striatal dopamine (DA) receptors of the D2 type and cortical [3H]5-hydroxytryptamine (5-HT)-binding sites. It is suggested that these results represent a down regulation of activity at striatal DA and cortical 5-HT synapses, since they now may operate at higher concentrations of transmitters. The reduced affinity may be caused by the physicochemical properties of toluene leading to changes in membrane fluidity and thus in the regulation of DA and 5-HT receptors in the local circuits of the striatum and the dorsal cerebral cortex.     

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.     

(3)H]-8-OH-DPAT binding in the rat brain raphe area: involvement of 5-HT(1A) and non-5-HT(1A) receptors

1. The 5-HT(1A) agonist 8-OH-DPAT has been shown to have additional 5-HT uptake inhibiting properties. The present work was undertaken to examine further the binding of [(3)H]-8-OH-DPAT in the raphe area of the rat brain, a region rich in 5-HT(1A) receptors and 5-HT uptake sites. 2. 5-HT inhibited [(3)H]-8-OH-DPAT binding in a biphasic manner (pK(i1): 8.82+/-0.01, pK(i2): 6.07+/-0.05, n=4) with the low affinity site representing 36+/-4% of the total population. A biphasic inhibition curve was found also with the 5-HT(1A) antagonist, WAY 100635 (pK(i1): 8.65+/-0.17, pK(i2): 4.26+/-0.38, n=3). In the presence of 1 microM WAY 100635 to mask 5-HT(1A) receptors, 5-HT inhibited [(3)H]-8-OH-DPAT binding in a monophasic manner (pK(i): 6.04+/-0.07, n=3). 3. The affinities of various compounds for sites labelled by [(3)H]-8-OH-DPAT in the presence of 1 microM WAY 100635 and for sites labelled by [(3)H]-citalopram (a selective 5-HT uptake inhibitor) were determined. There was a significant correlation between pK(i) values at 5-HT uptake sites and at non-5HT(1A) sites labelled by [(3)H]-8-OH-DPAT (r=0.80, P<0. 001, n=17), suggesting these latter sites to be 5-HT uptake sites. 4. Whereas the affinities of R(+) and S(-) enantiomers of 8-OH-DPAT for the 5-HT uptake site are similar, R(+)8-OH-DPAT has 10 times higher affinity for the non-5-HT(1A) site than S(-)8-OH-DPAT and was considered as an outlier in the correlation. It is suggested that [(3)H]-8-OH-DPAT labels other, as yet unknown binding sites in the raphe.     

Actions of 5-hydroxytryptamine and 5-HT1A receptor ligands on rat dorso-lateral septal neurones in vitro.

1. The actions of 5-hydroxytryptamine (5-HT) and some 5-HT1A receptor ligands on neurones in the rat dorso-lateral septal nucleus were recorded in vitro by intracellular recording techniques. 2. In the presence of tetrodotoxin (1 microM) to block any indirect effects, bath application of 5-HT (0.3-30 microM) hyperpolarized the neurones in a concentration-dependent manner and reduced membrane resistance. The hyperpolarization did not exhibit desensitization and was sometimes followed by a small depolarization. 3. The 5-HT1A receptor ligands, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT) and buspirone but not the non-selective 5-HT1 receptor agonist, 1-m-trifluoromethylphenylpiperazine (TFMPP), also hyperpolarized the neurones. 4. 5-HT, 8-OH-DPAT and DP-5-CT appeared to act as full agonists whereas buspirone behaved as a partial agonist. The estimated EC50S were: DP-5-CT 15 nM, 8-OH-DPAT 110 nM, 5-HT 3 microM and buspirone 110 nM. 5. At a concentration of 3 microM, the putative 5-HT1A receptor antagonists, spiperone, methiothepin, NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine) and MDL 73005EF (8-[2-(2,3-dihydro-1,4-benzodioxin-2-yl-methylamino)ethyl]-8- azaspiro[4,5]decane-7,9-dione methyl sulphonate), produced a parallel rightward shift in the concentration-response curve to 5-HT with no significant reduction in the maximum response. The estimated pA2 values were: NAN-190 6.79, MDL 73005EF 6.59, spiperone 6.54 and methiothepin 6.17.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential vascular alpha1-adrenoceptor blocking properties of an array of 5-HT receptor ligands in the rat

This study set out to analyse the potential ability of some 5-hydroxytryptamine (5-HT) receptor ligands widely used in cardiovascular experimental models to interact with vascular alpha1-adrenoceptors in the pithed rat. These ligands included: methiothepin, methysergide and metergoline (5-HT(1)/5-HT2); WAY-100635, buspirone, ipsapirone and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (5-HT(1A)); GR127935 (5-HT(1B/1D)); ketanserin, ritanserin, spiperone and pizotifen (5-HT2); granisetron and metoclopramide (5-HT3); tropisetron (5-HT3/5-HT4); ergotamine (5-HT(1B/1D), 5-ht(5A/5B)); clozapine (5-HT6/5-HT7); as well as LY215840 and mesulergine (5-HT2/5-HT7). For this purpose, the increases in diastolic blood pressure produced by the selective alpha1-adrenoceptor agonist, phenylephrine, were analysed before and after the above antagonists or saline. The adrenoceptor antagonist properties of prazosin (alpha1) and yohimbine (alpha2) were also analysed for comparison. Thus, the phenylephrine-induced vasopressor responses were dose-dependently antagonised with the following apparent rank order of potency by: prazosin > or = methiothepin > ketanserin > clozapine > or = lisuride > buspirone; this potency correlates with the affinity of these compounds for alpha1-adrenoceptor binding sites. In contrast, the other compounds were either devoid of any blocking effect on--or even potentiated (i.e. lisuride, methysergide, 8-OH-DPAT, granisetron and GR127935)--the responses to phenylephrine. These results show that methiothepin, ketanserin, clozapine, lisuride and buspirone can block alpha1-adrenoceptors in the rat systemic vasculature.     

Effects of 6-methoxytetrahydro-beta-carboline on 5-hydroxytryptamine binding in rat brain

6-MeOTHBC competes for 5-HT binding sites in rat brain in-vitro and in-vivo. The beta-carboline is significantly more active at the type 1 [3H]-5-HT, than the type 2 [3H]spiperone receptors, in-vitro. Following injection, 6-MeOTHBC significantly decreases [3H]-5-HT binding in the cortex. The ineffectiveness on [3H]spiperone binding in-vivo corresponds with the low affinity in-vitro. The effect of 6-MeOTHBC on central 5-HT binding may be a significant aspect of its 5-HT-ergic activity.     

Electrophysiological evidence that spiperone is an antagonist of 5-HT1A receptors in the dorsal raphe nucleus

The neuroleptic spiperone, which binds to 5-HT1A, 5-HT2 and dopamine (DA) receptors, was studied for its effects on serotonin (5-HT) and DA neurons in dorsal raphe nucleus and substantia nigra pars compacta, respectively. We found that 1 mg/kg i.v. spiperone, but not LY53837 (a 5-HT2 antagonist), antagonized the inhibition induced by 5-HT1A agonists 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and buspirone in the dorsal raphe nucleus. Lower spiperone doses blocked DA receptors in substantia nigra pars compacta, but did not affect 5-HT neurons. Doses of 8-OH-DPAT completely silencing dorsal raphe neurons were ineffective in substantia nigra pars compacta. However, buspirone antagonized DA receptors in substantia nigra pars compacta with doses similar to those depressing dorsal raphe neurons. It is concluded that spiperone is an antagonist of 5-HT1A receptors in the dorsal raphe nucleus.     

Native rat hippocampal 5-HT1A receptors show constitutive activity

Previous studies have shown that human 5-hydroxytryptamine (5-HT)1A receptors stably expressed in transfected cell lines show constitutive G-protein activity, as revealed by the inhibitory effect of inverse agonists, such as spiperone, on basal guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding. In the present study, we evaluated the constitutive activity of native rat 5-HT1A receptors in hippocampal membranes. Using anti-Galphao-antibody capture coupled to scintillation proximity assay under low sodium (30 mM) conditions, we observed high basal [35S]GTPgammaS binding to Galphao subunits (defined as 100%). Under these conditions, 5-HT and the prototypic selective 5-HT1A agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin [(+)-8-OH-DPAT] both stimulated [35S]GTPgammaS binding to Galphao to a similar extent, raising binding to approximately 130% of basal with pEC50 values of 7.91 and 7.87, respectively. The 5-HT1A-selective neutral antagonist [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) could block these effects in a competitive manner with pKb values (5-HT, 9.57; (+)-8-OH-DPAT, 9.52) that are consistent with its pKi value at r5-HT1A receptors (9.33). In this native receptor system, spiperone and methiothepin reduced basal [35S]GTPgammaS binding to Galphao in a concentration-dependent manner to 90% of basal with pIC50 values of 7.37 and 7.98, respectively. The inhibition of basal [35S]GTPgammaS binding induced by maximally effective concentrations of spiperone (10 microM) or methiothepin (1 microM) was antagonized by WAY100,635 in a concentration-dependent manner (pKb, 9.52 and 8.87, respectively), thus indicating that this inverse agonism was mediated by 5-HT1A receptors. These data provide the first demonstration that native rat serotonin 5-HT1A receptors can exhibit constitutive activity in vitro.     

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.     

Central 5-HT1A receptors and the mechanism of the central hypotensive effect of (+)8-OH-DPAT, DP-5-CT, R28935, and urapidil

This study investigated the central hypotensive effects of drugs that possess a high affinity for central 5-hydroxytryptamine (5-HT1A) binding sites; (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), N,N-dipropylcarboxamidotryptamine (DP-5-CT), erythro-1-(1-[2(1,4-benzodioxan-2-yl)-2-hydroxyethyl]- 4-piperidyl)-2-benzimidazolinone (R28935) and urapidil proved to possess high affinity and selectivity for central 5-HT1A binding sites, labeled by [3H]8-OH-DPAT. (+)8-OH-DPAT (0.1-10 micrograms/kg) given through the left vertebral artery of chloralose-anesthetized cats, lowered blood pressure by a biphasic dose-response curve. When given systemically, 10- to 100-fold higher doses of (+)8-OH-DPAT were necessary to obtain the same hypotensive effect when compared with central administration. Besides 8-OH-DPAT, R28935, DP-5-CT and urapidil also lowered blood pressure by a central mechanism in doses that were ineffective when given systemically. The central hypotensive effect of 0.3 micrograms/kg (+)8-OH-DPAT, 3 micrograms/kg DP-5 CT, and 3 micrograms/kg R28935 could be blocked by 100 micrograms/kg (-)pindolol, indicating that central 5-HT1A receptors are involved. High doses of (+)8-OH-DPAT (3-10 micrograms/kg) can also lower blood pressure by activating central alpha 2-adrenoceptors. The hypotensive effect of 300 micrograms/kg urapidil given through the vertebral artery could not be blocked by (-)pindolol. These results indicate the involvement of central 5-HT1A receptors in the mechanism of the central hypotensive activity of (+)8-OH-DPAT, DP-5-CT, and R28935.     

Serotonin-2 receptor-mediated regulation of release of acetylcholine by minaprine in cholinergic nerve terminal of hippocampus of rat

5-Hydroxytryptamine (5-HT) inhibited the K+-induced release of [3H]acetylcholine [( 3H]ACh) from slices of the hippocampus of the rat, dose-dependently. Minaprine (3-(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine, Fig. 1) had no effect on the release of [3H]ACh. However, it inhibited the (formula; see text) Fig. 1. Chemical structure of minaprine dihydrochloride. attenuation of the release of [3H]ACh by 5-HT dose-dependently. The 5-HT2 receptor antagonists, mianserine, methysergide and spiperone, prevented the inhibitory effect of the 5-HT, as well as did minaprine. The attenuating effect of 5-HT was not mimicked by the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and was not prevented by a 5-HT1A and 5-HT1B mixed receptor antagonist, propranolol, or by the 5-HT3 receptor antagonists, cocaine and metoclopramide. Minaprine inhibited the bindings of [3H]5-HT, [3H]8-OH-DPAT and [3H]ketanserin in the hippocampus. The inhibitory effect of minaprine on the binding of [3H]ketanserin was more marked than on the binding of [3H]5-HT and [3H]8-OH-DPAT, and was non-competitive. The Ki value of minaprine for the binding of [3H]ketanserin was 2.9 microM. The inhibitory effect of 5-HT on the release of [3H]ACh was observed in the presence of tetrodotoxin. By electrolytic lesioning of the medial septum, the K+-induced release of [3H]ACh from the slices of hippocampus was significantly reduced and the release was no longer inhibited by 5-HT. The lesioning significantly decreased the binding of [3H]ketanserin in the hippocampus, with hardly any reduction in the binding of [3H]5-HT and [3H]8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site

The kinetic and pharmacological characteristics of the binding of [3H]5-HT (serotonin), [3H]8-OH-DPAT (8-OH-2-di-n-propylaminotetraline), [3H]LSD, [3H]ketanserin and [3H]mesulergine to membranes from frontal cortex, hippocampus and choroid plexus of pig brain were studied. The binding of these ligands to frontal cortex and hippocampus demonstrated the presence of 5-HT1 and 5-HT2 sites in both tissues, although hippocampus was richer in 5-HT1 (subtype 5-HT1A) sites. [3H]5-HT, [3H]mesulergine and [3H]LSD labeled the pig choroid plexus with high affinity. The pharmacological profiles of [3H]5-HT and [3H]mesulergine binding to this tissue were closely comparable. Ligands reported as selective for 5-HT1A, 5-HT1B or 5-HT2 subtypes did not show high affinity for these binding sites. Therefore, these 5-HT binding sites in pig choroid plexus could be named 5-HT1C. Other drugs with a high affinity for these sites were methysergide and mianserine. In pig frontal cortex, [3H]5-HT labeled the different subtypes of 5-HT1 sites. In contrast, [3H]mesulergine bound in pig frontal cortex to a small population of sites with pharmacological properties similar to those of the choroid plexus 5-HT1C sites. Possible physiological functions in which these sites might be involved are discussed.