GAD(67): the link between the GABA-deficit hypothesis and the dopaminergic- and glutamatergic theories of psychosis.

Decreases in the 67 kDa isoenzyme of brain glutamic acid decarboxylase (GAD(67)) expression have been consistently found in patients with bipolar disorder and schizophrenia. In animals GAD(67) expression is diminished by chronic, but not acute stimulation of dopamine D(2) receptors and by short-term blockade of NMDA receptors. In contrast, chronic treatment with D(2) receptor antagonists enhances GAD(67) expression. Thus, antipsychotic treatment cannot explain the reduction in GAD(67) levels in patients with psychotic disorders. Rather, pathophysiological findings such as reduced viability of cortical glutamatergic neurones (in schizophrenia) or enhanced dopamine sensitivity (in bipolar disorder) might explain the observed reduction in GAD(67). Since reduction in GAD(67) expression leads to reduced levels of GABA, the GABAergic inhibitory control over glutamatergic cells is reduced. Psychosis could result from AMPA receptor activation caused by overactivity of the glutamatergic system. GAD(67) levels would thus be a surrogate marker for psychosis liability. Pharmacological principles that raise GAD(67) expression levels could represent novel targets for antipsychotic therapy.

Extended radioligand binding profile of iloperidone: a broad spectrum dopamine/serotonin/norepinephrine receptor antagonist for the management of psychotic disorders.

Iloperidone is a novel psychotropic compound currently undergoing Phase III trials. Its affinity for human dopamine and 5-HT(2A) and 5-HT(2C) receptors has been reported previously. This report presents the affinity of iloperidone for a largely extended number of human neurotransmitter receptors. In a few instances human receptors were not available and receptor studies were performed on tissues from laboratory animals. The present data, supplemented with those of, indicate that iloperidone displays high affinity (K(I) < 10 nM) for norepinephrine alpha(1)-adrenoceptors, dopamine D(3) and serotonin 5-HT(2A) receptors. Intermediate affinity (10-100 nM) was found for norepinephrine alpha(2C)-adrenoceptors, dopamine D(2A) and D(4) receptors and serotonin 5-HT(1A), 5-HT(1B), 5-HT(2C) and 5-HT(6) receptors. The affinity for all other receptors was below 100 nM, including norepinephrine alpha(2A), alpha(2B), beta(1), and beta(2), muscarine M(1)-M(5), histamine H(1), dopamine D(1) and D(5), CCK(A) and CCK(B), 5-HT(7), dopamine and norepinephrine transporters. Thus, iloperidone targets a selective set of dopamine, norepinephrine and serotonin receptor subtypes. The affinity for this particular set of receptors indicates that iloperidone has the potential to be a broad spectrum antipsychotic, with efficacy against positive, negative, depressive and cognitive symptoms of schizophrenia, and a low propensity to induce side effects.

The role of alpha2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent, clozapine, in the rat.

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat. 2.The close structural analogues of clozapine, loxapine (0.1 mg kg(-1) s.c.) and iso-clozapine (1 and 3 mg kg(-1) s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg(-1) s.c.) did not produce catalepsy, but at a dose of 1 mg kg(-1) significantly inhibited catalepsy induced by loxapine (0.3 mg kg(-1) s.c.). 3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D2/5-HT1A, D2/5-HT1B/1D and D2/alpha2-receptor affinity (KD) ratios: i.e. 30-100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D2 to 5-HT2A, 5-HT2C or D1 did not reflect the grouping of cataleptic and non-cataleptic compounds. 4. Co-treatment with the alpha2-adrenoceptor antagonists, yohimbine (1-10 mg kg(-1) s.c.), RX 821002 (1-10 mg kg(-1) s.c.) and MK-912 (0.3 and 1 mg kg(-1) s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg(-1)). Yohimbine (1-10 mg kg(-1) s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg(-1) s.c.). The alpha2-adrenoceptor antagonists had no effect per se. 5. Neither yohimbine (10 mg kg(-1)) nor RX821002 (3 mg kg(-1)) altered the cataleptic response to the D1 receptor antagonist, SCH 23390 (1 mg kg(-1) s.c.), while, like clozapine, both compounds abolished the response to the 5-HT2A receptor antagonist, MDL 100,151 (3 mg kg(-1) s.c.). 6. The present data strongly implicate alpha2-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

Cataleptogenic effect of subtype selective 5-HT receptor antagonists in the rat.

5-HT receptor antagonists with selectivity for 5-HT1A WAY-100635 (N-[2-[-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xanecarboxamide), 5-HT1B GR 127935 (N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'(5-methyl-1,2, 4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide x HCl), 5-HT2C SB 200646A (N-(1-methyl-5-indolyl)-N'-(3-pyridyl)urea x HCl) and 5-HT2A (ketanserin, fananserin and MDL 100,151 ((+/-)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-pipe ridinemethanol) receptors were tested for cataleptogenic responses in rats. WAY-100635 (0.1-3 mg/kg, s.c.), ketanserin (0.1-3 mg/kg, s.c.), MDL 100,151 (0.3-3 mg/kg, s.c.) and fananserin (RP 62203; 3 mg/kg, s.c.) induced a significant catalepsy. GR 127935 (1 mg/kg, s.c.), SB 200646A (without effect per se at 10 mg/kg, s.c.) and MDL 100,151 (0.3 mg/kg, s.c.) did not inhibit the cataleptic response to the dopamine D2 receptor antagonist, loxapine (0.3 mg/kg, s.c.). Catalepsy induced by MDL 100,151 (3 mg/kg) was blocked by co-treatment with clozapine, but not by SB 200646A (both at 10 mg/kg, s.c.). Although clozapine displays significant affinity to 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C receptors, the present results suggest that blockade of these receptors is not responsible for clozapine's anticataleptic activity.

Clozapine inhibits catalepsy induced by olanzapine and loxapine, but prolongs catalepsy induced by SCH 23390 in rats.

Loxapine (0.3 mg/kg s.c.), olanzapine (10 mg/kg s.c.) and SCH 23390 (R-(+)-chloro-2, 3, 4, 5-tetrahydro-3-methyl-5-phenyl-1-H-3-benzazepine; 1 mg/kg, s.c.), but not clozapine (10 mg/kg, s.c.), induced catalepsy in rats. Co-administration of clozapine (1, 3 and 10 mg/kg s.c.) dose-dependently inhibited loxapine-induced catalepsy. Clozapine (10 mg/kg s.c.) also prevented the induction of catalepsy by olanzapine. In addition, clozapine abolished the catalepsy induced by loxapine when it was administered after the response had fully developed. In contrast, the duration of SCH 23390-induced catalepsy was prolonged by clozapine, indicating that its anti-catalepsy effects against olanzapine and loxapine are unlikely to be caused by muscle relaxation, sedation or stimulation. Since SCH 23390-induced catalepsy is reported to be blocked by scopolamine, dizocilpine (MK-801) or 8-hydroxy-dipropylamino-tetralin, it is unlikely that muscarinic blockade, NMDA ion channel blockade and 5-HT1A receptor agonism, respectively, are involved in clozapine's action, but the mechanism by which clozapine exerts this anti-cataleptic effect remains unknown.

Hypersensitivity to meta-chlorophenylpiperazine (mCPP) in migraine and drug withdrawal.

Administration of a bolus dose of mCPP, a 5-HT2C receptor agonist, to rats provokes endocrine and behavioural effects that are reminiscent of some of the symptoms of human depression. Rats exposed to chronic mild stress (which is also a key factor in the precipitation of human depression) were hypersensitive to mCPP, whilst chronic treatment with antidepressant serotonin re-uptake inhibitors suppressed the responsiveness to mCPP. Similarities also exist with respect to withdrawal reactions following chronic alcohol or benzodiazepine abuse. In humans, a bolus dose of mCPP can cause alcohol craving (in abstinent alcoholics) and migraine (in susceptible persons), suggesting that there is a 5-HT2C receptor hyperresponsiveness in these conditions also. It is hypothesized that chronic treatment with SSRI's can prevent migraine attacks and drug craving.

Effects of ergotamine and dihydroergotamine on 5-hydroxytryptamine-2A receptors in the isolated rat aorta.

Ergotamine contracted isolated rat aorta rings with an intrinsic activity of 50% of that of 5-hydroxytryptamine. (5-HT, 0.1 mmol/l). Dihydroergotamine did not contract the tissue, but insurmountably blocked contraction in response to ergotamine and 5-HT. The 5-HT2A receptor antagonist, ketanserin (0.1 mumol/l), inhibited ergotamine (pKB 8.0) and 5-HT (pKB 8.1) induced contractions. These results indicate that in the rat aorta ergotamine is a partial 5-HT2A receptor agonist, whilst dihydroergotamine is an insurmountable 5-HT2A receptor antagonist. The present data could explain why ergotamine displays more cardiovascular and uterotonic side effects than dihydroergotamine.

Activation of meningeal 5-HT2B receptors: an early step in the generation of migraine headache?

Several pharmaceuticals are frequently dispensed to prevent or reduce the occurrence of migraine attacks. The prophylactic effect of these drugs has been suggested to be caused through blockade of serotonin (5-HT) receptors of type 5-HT2B or 5-HT2C. To elucidate which of these receptors is involved, we first used radioligand binding assays to determine the pharmacological profile of the human and rat-5-HT2B receptor. Furthermore, the potency of drugs used in migraine prophylaxis to stimulate or inhibit 5-HT2B or 5-HT2C receptor-mediated potency of drugs used in migraine prophylaxis to stimulate or inhibit 5-HT2B or 5-HT2C receptor-mediated phosphatidyl inositol hydrolysis was measured. All these drugs were found to block both human receptors. Correlation of the receptor affinities with the potencies used in migraine prophylaxis showed significant correlations, which were better for the 5-HT2B (P = 0.001) than for the 5-HT2C receptor (P = 0.005). Migraine headache is thought to be transmitted by the trigeminal nerve from the meninges and their blood vessels. Using the reverse transcription-polymerase chain reaction, the expression patterns of all cloned G-protein-coupled serotonin receptors were analysed in various human meningeal tissues. All tissues expressed 5-HT1Dbeta, 5-HT2A, 5-HT2B, 5-HT4 and 5-HT7 mRNAs. Only trace amounts of 5-HT2C receptor mRNA were found. With organ bath experiments we showed that the 5-HT2B receptor stimulated the relaxation of the pig cerebral artery via the release of nitric oxide. Our data support the hypothesis that 5-HT2B receptors located on endothelial cells of meningeal blood vessels trigger migraine headache through the formation of nitric oxide.

SDZ GLC 756, a novel octahydrobenzo[g]quinoline derivative exerts opposing effects on dopamine D1 and D2 receptors.

SDZ GLC-756, a novel octahydrobenzo[g]quinoline derivative, is equipotent in displacing [3H]SCH23390 from dopamine D1 receptors and [3H]205-501 from dopamine D2 receptor binding sites. It blocks dopamine sensitive adenylate cyclase with the same potency as SCH23390, indicating antagonist properties at dopamine D1 receptors. On the other hand, SDZ GLC 756 inhibits electrically evoked acetylcholine release from rat striatal slices with the same potency as the selective dopamine D2 receptor agonist bromocriptine. This effect is blocked by spiperone suggesting that it is mediated by dopamine D2 receptor activation. The opposing action of SDZ GLC 756 on dopamine D1 and D2 receptors is also evident in vivo. SDZ GLC 756, like SCH23390, blocks apomorphine-induced rearing in mice. On the other hand, it inhibits prolactin secretion and produces circling in unilateral 6-OHDA-lesioned rats, which is compatible with stimulant properties at dopamine D2 receptors. This drug might be a new tool to study linkage between dopamine D1 and D2 receptors.

RU 24969-induced locomotion in rats is mediated by 5-HT1A receptors.

The locomotor response to RU 24969 (5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl) 1H-indole; 10 mg/kg, s.c.), a preferential 5-HT(1B) receptor agonist, was investigated in the rat, using two novel 5-HT(1A) receptor antagonists, WAY-100635 (0.3 mg/kg and 1 mg/kg, s.c.) and SDZ 216-525 (0.3 mg/kg, s.c.), and the novel 5-HT(1B)/5-HT(1D) receptor antagonist, GR 127935 (1 mg/kg, s.c.). The antagonists per se did not alter spontaneous locomotion. Both selective 5-HT(1A) receptor antagonists blocked RU 24969-induced hyperlocomotion, whilst the 5-HT(1B) receptor antagonist was without effect. These results suggest that RU 24969-induced hyperlocomotion in the rat is mediated by 5-HT(1A) receptors.

Evidence for a 5-HT1D receptor-mediated hypothermic effect of the alpha 1-adrenoceptor agonist, SDZ NVI-085, in guinea-pigs.

The alpha 1-adrenoceptor agonist, SDZ NVI-085 ((-)-(4aR,10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4- methyl-9-(methylthio)-2H-naphth[2,3-b]-1,4-oxazine.HCl; 1 mg/kg i.p.), decreased body temperature of guinea-pigs. Two 5-HT1D receptor antagonists, GR127935 (N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl- 1,2,4-oxadiazol-3yl)[1,1-biphenyl]-4-carboxamide) and PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine; both compounds at 1 mg/kg i.p., -30 min) blocked this response, whilst the alpha 1-adrenoceptor blocker prazosin (1 mg/kg i.p.) and the 5-HT1A receptor antagonist, SDZ 216-525 (methyl 4-(-[4-(1,1m3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl ]-1-piperazinyl)1H- indole-2-carboxylate; 1 mg/kg i.p.) were inactive. Another alpha 1-adrenoceptor agonist, St 587 (2-(2-chloro-5-trifluoromethylphenylimino)-imidazoline; 1 mg/kg i.p.) did not alter body temperature. SDZ NVI-085-induced hypothermia in guinea-pigs is probably mediated by 5-HT1D receptors.

Expression of serotonin receptor mRNAs in blood vessels.

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1D beta, 5-HT2A, 5-HT2B, 5-HT4, and 5-HT7 receptor mRNA to different extents. New splice variants of the porcine 5-HT4 receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT1D beta, 5-HT2B, and 5-HT4 receptor mRNA, whereas in smooth muscle cells 5-HT1D beta, 5-HT2A, 5-HT7, and in some experiments 5-HT2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.

(+)-cis-4,5,7a,8,9,10,11,11a-octahydro-7H-10-methylindolo[1,7- bc][2,6]-naphthyridine: a 5-HT2C/2B receptor antagonist with low 5-HT2A receptor affinity.

The indolonaphthyridine 8 is described as a selective 5-HT2C/2B vs 5-HT2A receptor antagonist. The compound was synthesized in seven steps starting from indoline and isonicotinic acid chloride. The key step is a photocyclization of the indolinyl tetrahydropyridinocarbamic acid ethyl ester 4 to the cis-octahydroindolo[1, 7-bc][2,6]naphthyridinecarbamic acid ethyl ester 5. The synthesis was accomplished by reduction with aluminum hydride and racemic resolution. The indolonaphthyridine 8 exerted the binding profile of a selective 5-HT2C receptor ligand (pKD 7.8) and behaved as an antagonist on the 5-HT-induced accumulation of inositol phosphates in pig choroid plexus cells (pKB 7.13). Compound 8 dose-dependently inhibited the ACTH response to MK-212 in rats and the MK-212-induced hypophagic effect with an ID50 value of 0.3 mg/kg sc. Compound 8 acted as a 5-HT2B receptor antagonist at the rat stomach fundus with a pKB value of 7.34.

5-Hydroxytryptamine (5-HT) and the initiation of migraine: new perspectives.

The hypothesis that 5-hydroxytryptamine (5-HT) acting through 5-HT2C receptors is a key factor in the initiation of migraine has been re-evaluated in the light of recent basic and clinical scientific developments. The key findings are that nitric oxide is an important trigger for migraine, that 5-HT2B/5-HT2C receptors are present on endothelial cells and trigger nitric oxide release when activated and that supersensitivity of the 5-HT2B/5-HT2C receptor is a neurochemical feature predisposing to headache. Taken together the data bring new perspectives to the role of 5-HT acting through 5-HT2C (or closely similar) receptors in the initiation of migraine.

Is migraine prophylactic activity caused by 5-HT2B or 5-HT2C receptor blockade?

It has been suggested that activation of 5-HT2C receptors is involved in the initiation of a migraine attack. The 5-HT2C receptor and the newly cloned rat fundus 5-HT2B receptor show close pharmacological and structural resemblance. Antagonist pA2 values from the rat stomach and pKD values from a 5-HT2C receptor binding assay correlated both highly significantly (p < 0.005) with the daily dose of eight migraine prophylactic compounds. Although the small difference in antimigraine potency between the enantiomers of propranolol agrees with the lack of stereo-selectivity found on the rat fundus 5-HT2B receptor but not with the 5-HT2C receptor, the evidence available does not allow one to distinguish between 5-HT2C and 5-HT2B receptor blockade as possible mechanisms for prophylactic activity.

Cloning and functional characterization of the rat stomach fundus serotonin receptor.

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 x 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane-spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin-stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5-HT1C or 5-HT2 receptors.

5-HT in nervous system disease and migraine.

New developments defining the relationship between 5-hydroxytryptamine (5-HT; serotonin)1B and 5-HT1D receptors are reviewed and a novel pain control system involving spinal 5-HT3 receptors is described. The emerging roles of 5-HT receptor mechanisms in migraine and in the craving for alcohol are discussed.

Centrally acting alpha 1-adrenoceptor agonists based on hexahydronaphth[2,3-b]-1,4-oxazines and octahydrobenzo[g]quinolines.

Centrally acting alpha 1-agonists may be of therapeutic value in dementias and other CNS disorders characterized by symptoms of noradrenergic insufficiency. Therefore, on the basis of known peripherally acting alpha 1-agonists two new groups of centrally acting alpha 1-agonists with improved lipophilicity, the hexahydronaphth[2,3-b]-1,4-oxazines type A and the octahydrobenzo[g]quinolines type B were designed. The N-methylated derivatives 14 and 33 demonstrate potent, direct agonistic activity at postjunctional alpha 1-receptors. Ring substituent alterations in compounds of type A and B change the potency of compounds on the rabbit ear artery by over 3 orders of magnitude (pD2 = 5.35-8.40). The efficacy of these compounds varies from 42 to 110%. Those alpha 1-agonists which were selective in the pithed rat increase vigilance in rats. Compound 14 was found to be a centrally acting alpha 1-agonist with good tolerability in different animal species and in healthy volunteers. Furthermore, 14 selectively stimulates the breakdown of phosphatidylinositol in rat cerebral cortex slices. In vivo, the compound reverses behavioral deficits in animals which received noradrenergic lesions following DDC or DPS4 treatment. Oxazine 14 and its close derivatives are by far more lipophilic than commonly known alpha 1-agonists. This is demonstrated in a ClogP-PROBIS plot.

Agonist effects at putative central 5-HT4 receptors in rat hippocampus by R(+)- and S(-)-zacopride; no evidence for stereo-selectivity.

The EEG of halothane anaesthetized rats was recorded from an electrode implanted into the hippocampus. In the present study the effect of R(+)- and S(-)-zacopride, administered intra-cerebroventricularly, on different frequency bands of the EEG was investigated. Both enantiomers induced similar dose-dependent (5-20 micrograms) increases in all frequency bands. The effects of R(+)- and S(-)-zacopride were inhibited by pretreatment with a high dose of ICS 205-930 (1 micrograms i.c.v.), which suggests the involvement of 5-HT4 receptors. The lack of stereo-selectivity of the zacopride enantiomers is in contrast to observations made in in vitro studies.