Mammalian GFRalpha -4, a divergent member of the GFRalpha family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin.

Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.

Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions.

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.

Agonistic properties of alniditan, sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines.

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT1B/5-HT1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT1B receptor expressed at 200 fmol mg(-1) protein (Bmax) in non-induced L929sA cells, at 740 fmol mg(-1) protein in HEK 293 and at 2300 fmol mg(-1) protein in mIFNbeta-induced L929sA cells, and on the human cloned 5-HT1D receptor expressed in C6 glioma cells (Bmax 780 fmol mg(-1) protein). 2. Sodium butyrate treatment increased the expression level of human (h)5-HT1B receptors in HEK 293 cells and h5-HT1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [3H]-5-HT and [3H]-alniditan were unaffected. 3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the hS-HT1B receptor (IC50 values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and hS-HT1D receptors (IC50 values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin). 4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT1B receptor, and twice as potent at the h5-HT1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the hS-HT1D receptor.

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan.

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.

Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding.

Risperidone and its active metabolite 9-OH-risperidone were compared to reference antipsychotic drugs (haloperidol, pipamperone, fluspirilene, clozapine, zotepine) and compounds under development (olanzapine, seroquel, sertindole, ORG-5222, ziprasidone) for in vitro binding to neurotransmitter receptors in brain tissue and on membranes of recombinant cells expressing cloned human receptors and for in vivo occupancy of neurotransmitter receptors in rat and guinea-pig brain following acute treatment (2 h., s.c.). An ex vivo autoradiography technique was applied to determine the receptor occupancy by the drugs administered in vivo. Of particular interest are the central 5HT2A receptors and D2-type receptors. Predominant 5HT2A receptor antagonism is supposed to add to an atypical profile of the antipsychotics (treatment of the negative symptoms, low incidence of extrapyramidal side effects). D2 antagonism is required the treatment of positive symptoms. A contribution of the new dopamine receptor subtypes D3 and in particular D4 receptors has been proposed. In vitro, all compounds, except the 'typical' antipsychotics haloperidol and fluspirilene, showed higher affinity for 5HT2A than for D2 receptors. Subnanomolar affinity for human 5HT2A receptors was observed for ORG-5222, sertindole, risperidone, 9-OH-risperidone and ziprasidone. Fluspirilene, ORG-5222, haloperidol, ziprasidone, risperidone, 9-OH-risperidone and zotepine displayed nanomolar affinity for human D2 receptors. Sertindole and olanzapine were slightly less potent. Pipamperone, clozapine and seroquel showed 2 orders of magnitude lower D2 affinity in vitro. Clozapine, but even more so pipamperone, displayed higher affinity for D4 than for D2 receptors. For most other compounds, D4 affinity was only slightly lower than their D2 affinity. Seroquel was totally devoid of D4 affinity. None of the compounds had nanomolar affinity for D1 receptors; their affinity for D3 receptors was usually slightly lower than for D2 receptors. In vivo, ORG-5222, risperidone, pipamperone, 9-OH-risperidone, sertindole, olanzapine, zotepine and clozapine maintained a higher potency for occupying 5HT2A than D2 receptors. Risperidone and ORG-5222 had 5HT2A versus D2 potency ratio of about 20. Highest potency for 5HT2A receptor occupancy was observed for ORG-5222 followed by risperidone and olanzapine. Ziprasidone exclusively occupied 5HT2A receptors. ORG-5222, haloperidol, fluspirilene and olanzapine showed the highest potency for occupying D2 receptors. No regional selectivity for D2 receptor occupancy in mesolimbic versus nigrostriatal areas was detected for any of the test compounds. Risperidone was conspicuous because of its more gradual occupancy of D2 receptors; none of the other compounds showed this property. The various compounds also displayed high to moderate occupancy of adrenergic alpha 1 receptors, except fluspirilene and ziprasidone. Clozapine, zotepine, ORG-5222 and sertindole occupied even more alpha 1 than D2 receptors. Clozapine showed predominant occupancy of H1 receptors and occupied cholinergic receptors with equivalent potency to D2 receptors. A stronger predominance of 5HT2A versus D2 receptor occupancy combined with a more gradual occupancy of D2 receptors differentiates risperidone and its 9-OH-metabolite from the other antipsychotic compounds in this study. The predominant 5HT2A receptor occupancy probably plays a role in the beneficial action of risperidone on the negative symptoms of schizophrenia, whereas maintenance of a moderate occupancy of D2 receptors seems adequate for treating the positive symptoms of schizophrenia. A combined 5HT2A and D2 occupancy and the avoidance of D2 receptor overblockade are believed to reduce the risk for extrapyra

Genomic cloning, heterologous expression and pharmacological characterization of a human histamine H1 receptor.

A human histamine H1 receptor gene lacking introns was isolated by screening a human genomic library with a bovine histamine H1 receptor probe. The deduced protein of 487 amino acids showed characteristic properties of G-protein-coupled receptors. The coding region was subcloned into the expression vector pSVL (Pharmacia), and the resulting construct transfected into COS-7 cells. Binding studies with [3H]pyrilamine on membranes from transfected cells revealed saturable specific binding with a KD of 1.2 nM and a Bmax of 3400 fmol/mg protein. Binding affinities of histamine and known histamine antagonists were similar to those for histamine H1 receptors in guinea-pig cerebellum. In transfected COS-7 cells, histamine induced inositol phosphate formation, that was inhibitable by pyrilamine.

Activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists at cloned human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells.

The activity of serotonin (5-HT) receptor agonists, partial agonists and antagonists, and various other neurotransmitter receptor antagonists at human 5-HT1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells was investigated. 5-HT1A receptor-mediated inhibition of adenylate cyclase was studied by measuring inhibition of cAMP accumulation, induced by forskolin. At 100 microM forskolin produced a 100-fold increase in cAMP formation: 5-HT concentration dependently inhibited the cAMP formation; maximal inhibition was attained at 1 microM 5-HT and represented 90% of the stimulated cAMP formation. Full inhibition was observed with 5-HT1A receptor agonists: N,N-dipropyl-8-hydroxy-2-aminotetralin (8-OH-DPAT) and flesinoxan, and non-selective 5-HT receptor agonists: d-lysergic acid diethylamide (d-LSD), RU 24,969, bufotenine, methysergide and tryptamine. The rank order of potency of the compounds for inhibiting the cAMP formation corresponded to the rank order of the binding affinities of the drugs for the 5-HT1A receptor. Partial inhibition was obtained with submicromolar concentrations of buspirone, spiroxatrine and ipsapirone. A slight inhibition was observed with 1 microM 5-HT receptor agonist CP 93129 and 1 microM 5-HT receptor antagonists mesulergine and BW-501. No inhibition was found with: the 5-HT receptor agonists quipazine, sumatriptan and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); the 5-HT receptor antagonist ICS-205,930; and other neurotransmitter receptor antagonists such as pindolol, CGP 20712-A, prazosin, sulpiride and pyrilamine. Spiperone and pindolol fully antagonized the agonist-mediated inhibition of forskolin-stimulated cAMP formation. Partial inhibition of the agonist-mediated inhibition of forskolin-stimulated cAMP formation was apparent with 1 microM ocaperidone and 1 microM ipsapirone. It can be concluded that HeLa cells, permanently expressing human 5-HT1A receptors, are a valid cellular system for studying the negative coupling of 5-HT1A receptors to adenylate cyclase and the action of compounds thereupon.

Human beta 1- and beta 2-adrenergic receptor binding and mediated accumulation of cAMP in transfected Chinese hamster ovary cells. Profile of nebivolol and known beta-adrenergic blockers.

The interaction of nebivolol and its SRRR and RSSS enantiomers, and of known beta-adrenergic blockers, with human beta 1- and beta 2-adrenergic receptors expressed separately in Chinese hamster ovary cells in culture (CHO-Hu beta 1 and CHO-Hu beta 2), was investigated. We studied [3H]CGP-12177 binding to the intact cells and the accumulation of cAMP induced by isoproterenol. Each of the receptor subtypes displayed saturable [3H]CGP-12177 binding on intact cells with sub-nanomolar affinity. The density of beta 1- and beta 2-adrenergic receptor sites was 1.1 x 10(6) receptor binding sites per CHO-Hu beta 1 cell and 0.2 x 10(6) receptor binding sites per CHO-Hu beta 2 cell, respectively. The beta-adrenergic antagonists CGP 20712-A, ICI 118-551 and propranolol showed the same binding properties as beta-adrenergic receptors in previously described tissues or cells. The potencies of these compounds in inhibiting beta-adrenergic receptor mediated accumulation of cAMP corresponded well with their binding affinities. d-Nebivolol (SRRR) and nebivolol showed combined high affinity and selectivity for inhibition of beta 1-adrenergic receptor coupled accumulation of cAMP in CHO-Hu beta 1 cells (0.41 and 0.42 nM for d-nebivolol and nebivolol, respectively). l-Nebivolol (RSSS) was 1460 times less potent than d-nebivolol in CHO-Hu beta 1 cells. The binding affinities of d-nebivolol and nebivolol for human beta 1-adrenergic binding sites correlated well with their potencies in inhibiting beta 1-adrenergic receptor coupled accumulation of cAMP. CHO cells transfected with human beta 1- and beta 2-adrenergic receptors are a valid model system for studying the interaction of compounds with human beta-adrenergic receptors.

Rapid desensitization and resensitization of 5-HT2 receptor mediated phosphatidyl inositol hydrolysis by serotonin agonists in quiescent calf aortic smooth muscle cells.

Agonist regulation of 5-hydroxytryptamine2 (5-HT2) receptors was studied in calf aortic smooth muscle cultures incubated in a quiescent, defined synthetic medium that does not stimulate cell proliferation, but that provides cells with supplements that maintain cell viability. In these cells, 5-hydroxytryptamine (5-HT)-induced [3H]inositol phosphates accumulation showed the characteristics of a 5-HT2 receptor coupled transducing system according to the inhibition of the response by 5-HT2 antagonists at nanomolar concentrations. The 5-HT2 receptor coupled response became rapidly desensitized during continued incubation with 5-HT and 1-(2,5-dimethoxy-4-methylphenyl)-2- aminopropane (DOM); nearly full desensitization was obtained in two hours with 10 microM 5-HT and DOM pretreatment. The recovery of the response had a half-live of 5 hours after 2 hours pretreatment and of 9.5 to 12.5 hours after 24 to 96 hours agonist pretreatment. The DOM-induced desensitization of the 5-HT2 receptor coupled response was fully blocked by 0.1 microM cinanserin. Cinanserin alone did not induce desensitization or up-regulation of the 5-HT2 receptor coupled response at 0.1 microM. It may be that the down-regulation of central 5-HT2 receptors by antagonists in vivo is a heterologous process due to mediators which are triggered by 5-HT2 antagonistic action.

The dissociation rate of unlabelled drugs from receptor sites: a poorly investigated, yet important aspect in receptor research. Studies on the serotonin-S2 receptor.

The labelling by 3H-spiperone of serotonin-S2 receptors in rat frontal cortex tissue adsorbed to glass fibre filters was investigated. For 12 unlabelled serotonin antagonists the dissociation time from serotonin-S2 receptors was measured using rat frontal cortex tissue preparations adsorbed to glass fibre filters. The dissociation half-time varied from 4.8 min for pipamperone to 160 min for ritanserin. The drug-receptor dissociation time was not related to a particular class of chemical structure, or to the lipophilicity or the acid dissociation constant of the drugs. The essential requirement of experimental determination of the drug-receptor dissociation time for each drug individually is illustrated. The possible applications of the knowledge of the drug-receptor dissociation time in in vitro and in vivo receptor studies, in pharmacological and pharmacokinetic studies and in drug design and receptor modelling is discussed. For various serotonin-S2 antagonists, the type of inhibition produced by the drug on 3H-ketanserin binding to serotonin-S2 receptors was determined using suspensions of rat frontal cortical tissue. The observed patterns of inhibition were clearly related to the drug-receptor dissociation times: rapidly dissociating drugs produced competitive inhibition, drugs with dissociation half-times between 15-30 min produced mixed type inhibition, and the very slowly dissociating ritanserin produced non-competitive inhibition.

Identification of nonserotonergic [3H]ketanserin binding sites associated with nerve terminals in rat brain and with platelets; relation with release of biogenic amine metabolites induced by ketanserin- and tetrabenazine-like drugs.

In mammalian striatal tissue and cat platelets, [3H]ketanserin labels besides serotonin-S2 receptors nonserotonergic saturable binding sites. The sites have been distinguished and characterized in [3H]ketanserin binding assays by selective inhibition with tetrabenazine (Ki = 4 nM), a monoamine depleting agent. In rats, the nonserotonergic ketanserin sites were enriched in the striatum (KD = 12.4 +/- 0.3 nM, maximal number of binding sites = 53.2 +/- 11.8 fmol/mg of tissue at pH 7.7, 37 degrees C) and nucleus accumbens. The sites were decreased by 65 to 78% after 6-hydroxydopamine lesions, suggesting an association with dopaminergic nerve terminals. In in vitro superfusion experiments using [3H]dopamine, [3H]norepinephrine and [3H]serotonin loaded rat brain tissue and [3H]serotonin loaded human platelets, 5 min superfusion with 10(-6) M ketanserin, tetrabenazine and reserpine caused instantaneously a marked increase in tritium efflux. The effect was attenuated by the monoamine oxidase inhibitor, pargyline, in brain slices but not in platelets. High-performance liquid chromatography analysis of endogenous catecholamines, serotonin and metabolites in superfusates from striatal slices revealed that stimulation with these drugs provoked mainly release of 3,4-dihydroxybenzeneacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. Potencies of a series of ketanserin derivatives, benzoquinolizine derivatives and a variety of drugs affecting neurotransmission were assessed in the in vitro release test using [3H]dopamine loaded striatal slices, and in [3H]ketanserin binding assays to nonserotonergic sites in the striatum and to serotonin-S2 receptors in brain tissue. Activities of drugs in the release test correlated strongly with their binding affinities for nonserotonergic ketanserin sites (rs = 0.83, n = 30, P less than .001). High potency in the latter two tests was confined to few close structural congeners of ketanserin and tetrabenazine. Distinct structural activity relationships for interaction with nonserotonergic ketanserin sites and serotonin-S2 receptors were found. It was concluded that nonserotonergic ketanserin sites mediate release of oxidated metabolites of biogenic amines from nerve endings and of serotonin from platelets. Hence release of biogenic amine metabolites or of cytoplasmic amines is probably not a mere diffusion process but involves specific membranous molecules. Unlike tetrabenazine, ketanserin caused no obvious depletion of central catecholamine and indoleamine stores. Implications of these findings for the mechanism of action of the drugs are discussed.

Distinction between adrenergic and serotonergic receptor subtypes: specificity of drugs and absence of cooperative interactions between adrenergic and serotonergic receptor binding sites.

In light of observed amplificatory interactions between serotonergic and adrenergic stimuli in functional studies on vascular tissue and platelets, we investigated the distinction and possible interactions between alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic and 5-HT1A-, 5-HT1B-, and 5-HT2-serotonergic receptor binding sites. Therefore, the binding affinities of archetypes of adrenergic and serotonergic agonists and antagonists for the various receptors were measured. Only the alpha 1-blocker prazosin revealed great specificity for alpha 1-adrenergic receptors; the other investigated antagonists and agonists showed cross-reactivity with adrenergic and serotonergic receptors in various combinations. Using [3H]ketanserin binding to rat frontal cortex and [3H]prazosin binding to rat cortex tissue as models for 5-HT2-serotonergic and alpha 1-adrenergic receptors, respectively, we did not find cooperative effects of epinephrine on the binding of 5-hydroxytryptamine or ketanserin to 5-HT2 receptors nor of 5-hydroxytryptamine on the binding of epinephrine or prazosin to alpha 1-adrenergic receptors. It was concluded that the various adrenergic and serotonergic receptor subtypes (a) have distinct drug binding properties; (b) occur on various central and peripheral tissues; (c) co-occur on some tissues; (d) each subtype mediates several distinct functions; (e) distinct receptors may mediate similar functions; (f) the drug binding properties of a particular receptor remains the same in different tissues, but purported alpha 2-like receptors on platelets reveal some differences from alpha 2-receptors in the brain and other peripheral tissues; (g) the various receptor subtypes appear to be distinct molecular entities; and (h) in brain tissue there is no evidence for the occurrence of direct adrenergic-serotonergic receptor-receptor interactions at the level of the binding sites.

Receptor interactions of dopamine and serotonin antagonists: binding in vitro and in vivo and receptor regulation.

The advent of receptor binding techniques has provided new ways of studying the mechanism of action of drugs. In vitro radioligand binding is now currently applied to investigate the specificity or multiple action of compounds. By using the same technique, the binding affinity of a drug can be measured for a variety of neurotransmitter, drug, peptide and ion channel receptor binding sites, providing the drug's receptor binding profile (LEYSEN et al. 1981; LEYSEN 1984). However, in vitro receptor binding is only the initial step in the investigation of drug-receptor interactions. Investigations in vivo are required to allow evaluation of how and where a drug acts. In fact, the study of drug-receptor interactions comprises three main stages: (a) in vitro radioligand receptor binding; (b) in vivo receptor binding, providing information on the accessibility of the drugs to the receptors localized in various central and peripheral tissues, on the drug potency for occupying various receptors, on the duration of receptor occupation and on the relationship between the degree of receptor occupation and pharmacological effects; and (c) the study of receptor regulation: the effect of chronic drug treatment on receptor alterations compared with alterations in functional responses in vivo. In this article, we will illustrate the three stages of investigation of receptor interactions and discuss the relevance and importance of the findings, using as examples three drugs known in psychopharmacological research: (a) the neuroleptic haloperidol, a prototype of a dopamine D2 antagonist: (b) Setoperone, a potential antipsychotic agent with very potent serotonin S2 and moderate D2 antagonistic activity (CEULEMANS et al. 1985; LEYSEN et al. 1986); and (c) ritanserin, a potent and long-acting S2 antagonist (LEYSEN et al. 1985), which has revealed therapeutic activity in dysthymia and negative symptoms of schizophrenia (REYNTJENS et al. 1986; GELDERS et al. 1986). Particular attention will be paid to the problem of receptor regulation. We challenge the general applicability of the receptor regulation theory, which states that persistent receptor stimulation causes desensitisation and receptor downregulation, whereas chronic deprivation of receptor stimulation leads to supersensitivity and receptor upregulation. Recent research has revealed that the theory does not hold for S2 receptor alterations, which were found to downregulate following chronic receptor blockade.

Agonist-induced desensitization of 5-HT(2) receptors on cultured calf aortic smooth muscle cells.

[(32)P]Phosphatidic acid (PA)-formation was quantified in calf aortic smooth muscle cultures for measuring the activation of the signal transducing system coupled to the 5-hydroxytryptamine(2)-(5-HT(2)) receptor. [(32)P]PA-formation was increased upon stimulation of smooth muscle cells with serotonin (5-HT) and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), but not with the 5-HT(1) agonists N,N-dipropyl-8-hydroxy-2-aminotetralin and RU 24969. The potency of drugs to inhibit the 5-HT induced [(32)P]PA-formation closely corresponded to their binding affinity for 5-HT(2) receptors. 24-Hour treatment of smooth muscle cultures with 5-HT or DOM resulted in a substantial decrease of 5-HT induced [(32)P]PA-formation. In contrast to the anomalous 5-HT(2) receptor regulation in vivo, 5-HT(2) receptors on smooth muscle cells appeared to be desensitized by agonist treatment.

Opposite regulation of serotonin-S2 and dopamine-D2 receptors in rat brain following chronic receptor blockade.

Rats were chronically treated with setoperone, a mixed serotonin and dopamine antagonist. Alterations in serotonin-S2 and dopamine-D2 receptors in the brain and changes in behavioural responses to tryptamine and apomorphine were studied along with duration of treatment and drug withdrawal. As with neuroleptics, behavioural supersensitivity to apomorphine and increase in the number of striatal dopamine-D2 receptor sites were apparent after 2 days setoperone treatment, both effects were maximal with 14 days treatment and were maintained over more than 20 days drug withdrawal. In contrast to the changes in the dopaminergic system, the rats showed a decreased response to tryptamine and serotonin-S2 receptor sites in the frontal cortex were significantly reduced in numbers. Both effects developed in parallel over 14 days treatment and extinguished over 10 days drug withdrawal. KD-values of radioligand binding to dopamine-D2 and serotonin-S2 receptor sites were unchanged by the setoperone treatment. The concomitant development and extinction of the in vivo and in vitro effects suggests a causal relationship between them. Chronic treatment with a selective histamine-H1 antagonist (levocabastine) or the tranquilizer diazepam did not affect dopamine-D2 or serotonin-S2 receptor sites. These observations demonstrate that in contrast to the receptor regulation theory, serotonin-S2 receptors are down regulated following persistent receptor blockade. Implications for the clinical use of serotonin antagonists and possible molecular mechanisms involved in the receptor regulation have been discussed.

Down regulation of serotonin-S2 receptor sites in rat brain by chronic treatment with the serotonin-S2 antagonists: ritanserin and setoperone.

Ritanserin is a potent and selective serotonin-S2 antagonist which slowly dissociates from the receptor sites, while setoperone has potent serotonin and moderate dopamine antagonistic properties and dissociates rapidly from the receptor sites. Acute administration of ritanserin (1-10 mg/kg) produced a non-competitive inhibition of 3H-ketanserin binding, measured ex vivo in washed frontal cortex membranes, which lasted for 12 h. This is in accordance with the slow dissociation of the drug from the receptor sites. Setoperone (1-10 mg/kg orally) also produced a partially non-competitive inhibition of 3H-ketanserin binding in washed membranes, which is unlike its rapid dissociation. In contrast, there was no inhibition of dopamine receptor binding in washed striatal membranes. Chronic oral administration of 10 mg/kg X day of the drugs significantly reduced the Bmax values of 3H-ketanserin, without changing the KD value when drug-free periods were longer than 1 day. The maximum reduction following 25 days' treatment with 14 mg/kg ritanserin was 50% at 1 day drug-free; the Bmax values gradually returned to the control value in about 12 days. The receptor half-life was calculated to be 3.5 days and the receptor synthesis rate 4 fmoles/mg tissue X day. Ritanserin treatment did not alter radioligand binding to serotonin-S1, alpha 1-, alpha 2- and beta-adrenergic, dopamine-D2, benzodiazepine and substance P sites. Chronic treatment with setoperone at 10 mg/kg X day, orally, significantly reduced the Bmax value of 3H-ketanserin binding in frontal cortex but treatment with 1 mg/kg X day did not. In contrast, a dose-dependent increase in the number of striatal dopamine-D2 sites was observed, in accordance with the moderate dopamine-antagonistic properties of setoperone. Dopamine-D2 receptor up regulation up to 150% of control values, was maintained at the same level for 9 days, it started to decline 12 days after stopping drug treatment. Following chronic treatment and drug withdrawal for more than 1 day, ritanserin and setoperone levels in whole brain homogenates were below detection level (less than 1 ng/g). The similar reduction in the Bmax values of 3H-ketanserin binding following chronic treatment with the rapidly dissociating setoperone and the slowly dissociating ritanserin, the absence of effect on the KD value, the slow reappearance of the receptor sites and the opposite effect on serotonin-S2 and dopamine-D2 receptors with setoperone suggest that real serotonin-S2 receptor down regulation occurs following antagonist treatment. The findings illustrate the difference in receptor regulation between the serotonergic and the dopaminergic system.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptor-binding properties in vitro and in vivo of ritanserin: A very potent and long acting serotonin-S2 antagonist.

In vitro and in vivo receptor-binding properties of the new serotonin antagonist, ritanserin, are reported. In in vitro binding assays, ritanserin shows high affinity binding to serotonin-S2 sites in rat frontal cortex tissue: IC50 = 0.9 nM without drug preincubation and 0.3 nM with 30-min drug preincubation; IC50 values for histamine-H1, dopamine-D2, and adrenergic-alpha 1 and -alpha 2 sites were 39-, 77-, 107-, and 166-fold higher, and at up to 1 microM, the drug did not bind to serotonin-S1 sites. In in vitro assays, ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.(ABSTRACT TRUNCATED AT 400 WORDS)