Functional activation of G-proteins coupled with muscarinic acetylcholine receptors in rat brain membranes.

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes.

Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.

Activation of Gq proteins coupled with 5-HT2 receptors in rat cerebral cortical membranes assessed by antibody-capture scintillation proximity assay/[S]GTPγS binding.

BACKGROUND/AIMS: Functional activation of Gq coupled with 5-HT2 receptors was investigated in rat cerebral cortical membranes. METHODS: Antibody-capture scintillation proximity assay (SPA)/[(35)S]GTPγS binding with anti-Gαq antibody was performed. RESULTS: The specific [(35)S]GTPγS binding to Gαq was increased by 5-hydroxytryptamine (5-HT) in a concentration-dependent but unsaturable manner. The increase elicited by micromolar concentrations of 5-HT was inhibited completely by ketanserin, whereas it inhibited the response by submillimolar to millimolar concentrations of 5-HT only partially. Analysis of the concentration-dependent increases by 5-HT in the absence and presence of ketanserin, methiothepin, WAY100635, and pirenzepine clearly indicates that there are two distinct components of 5-HT-stimulated [(35)S]GTPγS binding, one of which is a pharmacologically relevant increase elicited by lower concentrations (-30 µmol/l) of 5-HT mediated through 5-HT2 receptors and the other is pharmacologically undefined stimulation by higher concentrations of 5-HT. When 5-HT and carbachol were added simultaneously, there was apparently lack of additivity. CONCLUSION: It is concluded that by means of antibody-capture SPA/[(35)S]GTPγS binding it is possible to detect two distinct components of 5-HT-elicited activation of Gq shared by M1 muscarinic receptors, one of which is mediated through 5-HT2 receptors and the other is derived from unknown origin in rat cerebral cortical membranes.

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes.

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [(35)S]GTPγS binding, using the anti-Gαq antibody sc-393. Increased specific [(35)S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gαq, selectively coupled with the M1 mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M1 mAChR agonists in the present study were generally lower than previously reported in M1-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [(35)S]GTPγS binding to Gαq was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M1-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the Gq-coupled M1 mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.

Group II metabotropic glutamate receptor-mediated activation of G-proteins in rat hippocampal and striatal membranes.

Stimulation of G-proteins coupled with metabotropic glutamate receptors (mGlu receptors) was investigated by means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay in rat hippocampal and striatal membranes. The endogenous ligand l-glutamate increased specific [(35)S]GTPγS binding in a concentration-dependent manner with a mean EC50 values of around 10µM in both brain regions, The maximal % increase over the respective basal binding was highest in cerebral cortex, intermediate in hippocampus, and lowest in striatum. The pharmacological profiles of the responses investigated with a series of glutamatergic agonists and antagonists clearly indicated that they were mediated through group II mGlu receptors, particularly mGlu2 subtype, in both brain regions. The pEC50 and relative %Emax values for a series of agonists were essentially identical in both brain regions that were also correlated with those previously reported in cerebral cortical membranes. The selective allosteric potentiator of mGlu2 receptor subtype, LY487379, potentiated the increasing effects of l-glutamate at a maximally effective concentration of 1mM on specific [(35)S]GTPγS binding, without altering the basal unstimulated binding. It is concluded that [(35)S]GTPγS binding assay is applicable to rat hippocampal and striatal membranes to detect functional activation of Gαi/o proteins coupled with mGlu2 receptors.

Muscarinic acetylcholine receptor-mediated activation of G(q) in rat brain membranes determined by guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding using an anti-G protein scintillation proximity assay.

In the present study, we performed antibody-capture guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) scintillation proximity assay (SPA), in which immuno-capture of Gα subunits following [(35)S]GTPγS binding was combined with SPA technology, in rat brain membranes. Preliminary experiments using a series of agonists and commercially available anti-Gα antibodies indicated the increase in specific [(35)S]GTPγS binding to Gα(q) determined with the anti-Gα antibody sc-393 and evoked by carbamylcholine chloride (CCh) was pharmacologically relevant. The experimental conditions were optimized as for the concentrations of GDP, MgCl(2), and NaCl, the dilution of the anti-Gα(q) antibody, and membrane protein contents incubated. Under the optimized conditions, CCh-stimulated specific [(35)S]GTPγS binding to Gα(q) in a concentration-dependent and saturable manner with an EC(50) of around 10 µM in all of the membranes prepared from rat hippocampus, cerebral cortex, and striatum. The maximum responses were varied according to the brain regions, with the rank order in magnitude of hippocampus > cerebral cortex > striatum. The addition of MT-7, a snake toxin with high selectivity for M(1) over the other muscarinic acetylcholine receptors (mAChRs) (M(2)-M(5)), almost completely extinguished CCh-stimulated [(35)S]GTPγS binding to Gα(q), even at a concentration as low as 1 nM. These results indicate that the functional coupling between M(1) mAChR and Gα(q) can be investigated in rat native brain membranes by means of antibody-capture SPA/[(35)S]GTPγS binding assay. The assay developed in the present study would provide a useful strategy for investigation of possible pathophysiological alterations in neuropsychiatric disorders such as Alzheimer's disease and schizophrenia as well as for drug discovery.

[Visual presentation of psychiatric clinical decision-making by "graphic assessment sheet for diagnoses and treatments"].

Psychiatrists often have to treat patients even when the clinical information is insufficient to make a definite diagnosis. This is the case especially when we are treating first-visit outpatients or inpatients who have just been admitted. One of the causes of information insufficiency is a delay in obtaining clinical information on the patient, and another is a lack of characteristic manifestations of the disease because of an immature developmental stage. Even in such situations, however, clinicians have to make reasonable judgements using the information that is available at that time. The framework for making judgements on such occasions, or "the framework of decision-making under imperfect-information conditions", is becoming more and more important in psychiatric clinical practice in Japan for the following reasons. First, team members in charge of a patient became very heterogeneous in terms of their career and motivation after the start of the new post-graduate clinical training system in Japan several years ago, resulting in a higher risk of miscommunication. Secondly, the need for precise explanation to patients and their families has become crucial in recent years as the result of various social changes. Ota T, one of the authors, once put forward the framework of decision-making under imperfect-information conditions on the basis of Bayesian statistics. In the present paper, in consideration of the above background, we devised a sheet for visualizing the above framework so that relevant staff could share the clinical decision-making process. Specifically, we visually arranged on a sheet of paper the components and variables of the framework, so that the staff could communicate with each other explicitly and precisely about the estimated probability of each possible disease, merits and demerits of each treatment option, etc. We employed the sheet on treating patients in our acute psychiatric ward, 2 of whom are presented in the paper. Discussions were made on the usefulness, limitations, and remaining problems.

Functional coupling between metabotropic glutamate receptors and G-proteins in rat cerebral cortex assessed by guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assay.

Stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding by l-glutamate was pharmacologically characterized in rat cerebral cortical membranes. Optimization of the experimental conditions with respect to the concentrations of GDP, MgCl(2) and NaCl in assay buffer prompted us to adopt the incubation of rat cerebral cortical membranes with 0.2 nM [(35)S]GTPγS at 30°C for 60 min. in the presence of 20 µM GDP, 5 mM MgCl(2) and 100 mM NaCl as a standard condition. Specific [(35)S]GTPγS binding was stimulated by l-glutamate in a concentration-dependent manner but not by ionotropic glutamate receptor agonists. The stimulatory responses were also elicited by many agonists for metabotropic glutamate (mGlu) receptor, with (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) being the most potent. l-glutamate-stimulated [(35)S]GTPγS binding was inhibited by several mGlu antagonists, with (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) being the most potent. The pharmacological properties of a series of agonists and antagonists indicated the involvement of group II mGlu receptors, especially mGlu2. Supportive of this notion was the finding that l-glutamate-stimulated specific [(35)S]GTPγS binding was augmented by 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulphonamide hydrochloride (LY487379), a reportedly selective allosteric positive modulator for mGlu2, by means of upward and leftward shift of the concentration-response curve. In addition, LY487379 per se stimulated [(35)S]GTPγS binding, though, through a mechanism different from the stimulation by l-glutamate. Pre-treatment of the membranes with N-ethylmaleimide (NEM) cancelled l-glutamate-stimulated [(35)S]GTPγS binding in a concentration- and incubation time-dependent manner. Taken altogether, l-glutamate-stimulated [(35)S]GTPγS binding serves as a useful functional assay for the activation of NEM-sensitive G(i/o) -mediated group II mGlu receptors in rat cerebral cortical membranes.

Angiotensin II type 1 receptor blockers improve insulin sensitivity in patients with schizophrenia being treated with olanzapine.

RATIONALE: Olanzapine (OLZ) is known to cause weight gain and metabolic disturbances, which may have serious implications with respect to medical comorbidities such as metabolic syndrome and insulin resistance. OBJECTIVES: The aim of this study was to evaluate the effects of two angiotensin II type 1 receptor blockers (ARBs) which are widely used as antihypertensive agents, valsartan (VAL) and telmisartan (TEL), on insulin resistance in patients with schizophrenia treated with OLZ. METHODS: Thirty inpatients with schizophrenia with OLZ monotherapy over 8 weeks participated in this study. To assess insulin resistance, the homeostasis model assessment of insulin resistance (HOMA-IR), fasting plasma glucose (PG) levels and immunoreactive insulin (IRI) levels were measured [HOMA-IR = fasting PG level (mmol/L) x fasting IRI level (µU/ml)/22.5]. VAL add-on treatment was performed in insulin-resistant patients (HOMA-IR > 1.6) for 12 weeks. After a 12-week VAL washout period, TEL add-on treatment was carried out for 12 weeks. The effects of ARBs on insulin resistance and other metabolic variables were assessed. RESULTS: In all 30 patients, both body mass index and abdominal circumference were strongly correlated with HOMA-IR. Twelve patients showed high HOMA-IR and were deemed to be insulin resistant. Add-on therapy of VAL and TEL resulted in a significant decrease in fasting IRI levels and HOMA-IR. No differences in any effects were observed between VAL and TEL. No adverse effects of either ARBs were observed in this study. CONCLUSIONS: ARBs for patients treated with OLZ improved insulin sensitivity and attenuated insulin resistance.

Pharmacological characterization of alpha2D-adrenergic receptor-mediated [35S]GTPgammaS binding in rat cerebral cortical membranes.

Using the rat cerebral cortical membranes that had once been frozen and stored at -80 degrees C, the stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding mediated through alpha(2)-adrenergic receptors (alpha(2)-ARs) was pharmacologically characterized. The stimulatory effects of (-)-noepinephrine ((-)-NE) and 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK-14,304) at maximally effective concentrations were prominent only in the presence of micromolar to submillimolar levels of GDP, with the greatest signal/noise ratio achieved at 100 and 30-100 microM GDP for (-)-NE and UK-14,304, respectively. The alpha(2)-AR-mediated [(35)S]GTPgammaS binding was also critically dependent on the presence of millimolar concentrations of MgCl(2) in assay medium. The maximum stimulation induced by UK-14,304 was equivalent to, or even greater than, that of an endogenous ligand (-)-NE at lower concentrations of NaCl, while it became a partial agonist with higher concentrations of NaCl. Concentration-response curves for several agonists revealed that the omission of NaCl from incubation buffer generally shifted the curves leftward and increased the relative efficacies as compared to the endogenous ligand. The pharmacological profile characterized with a series of adrenergic agonists and antagonists indicated that this response was derived from activation of G proteins coupled with alpha(2)-ARs, in particular alpha(2D)-AR subtype, though the possible involvement of alpha(2C)-ARs was not completely excluded. However, the stimulatory effects of several adrenergic compounds, such as rauwolscine and yohimbine, were ascribed to their agonist properties at 5-HT(1A) receptors. This method serves as a simple but useful strategy for investigating the functional interaction between alpha(2D)-ARs and their coupled G proteins in rat native cerebral cortical membranes, especially in the field of psychopharmacology and biological psychiatry.

5-HT1A receptor agonist properties of antipsychotics determined by [35S]GTPgammaS binding in rat hippocampal membranes.

1. 5-Hydroxytryptamine 1A (5-HT1A) receptors have attracted increasing attention as a promising target for antipsychotic therapy. Although many atypical antipsychotic drugs, including the prototype clozapine, have been reported to be partial agonists at 5-HT1A receptors, these results are often fragmental and derived mainly from experiments that used cultured cells. 2. In the present study, [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding assay in rat hippocampal membranes was applied to a series of antipsychotic drugs, especially atypical antipsychotics. 3. Most, but not all, of atypical antipsychotic drugs and the classical antipsychotic drug nemonapride behaved as partial agonists at 5-HT1A receptors with varied potencies and relative efficacies. The most potent compound was perospirone with a mean EC50 of 27 nmol/L, followed by aripiprazole (45 nmol/L) > ziprasidone (480 nmol/L) > nemonapride (790 nmol/L) > clozapine (3900 nmol/L) > quetiapine (26,000 nmol/L). The maximal percentage increases over the basal binding (%Emax) for these antipsychotic drugs were 30-50%, with the exception of perospirone (approximately 15%), whereas 5-HT stimulated the binding to a mean %Emax of 105%. 4. Increasing concentrations of the selective and neutral 5-HT1A antagonist WAY100635 shifted the concentration-response curve of nemonapride-stimulated [35S]GTPgammaS binding to the right and in parallel. 5. The relative efficacy or intrinsic activity of a compound was affected differently by the differing concentrations of guanosine diphosphate (GDP) in the assay buffer, which should be taken into consideration when determining the relative efficacies of these antipsychotics as 5-HT1A receptor agonists. 6. These results provide important information concerning the relevance of 5-HT1A receptor partial agonist properties in the treatment for schizophrenic patients with most, if not all, of atypical antipsychotic drugs.

Psychiatric problems of heart transplant candidates with left ventricular assist devices.

Most heart transplant candidates are equipped with left ventricular assist devices (LVADs). LVAD therapy is associated with characteristic psychiatric and psychosocial problems. To investigate the mental states of heart transplant candidates, psychiatric diagnosis, psychological or behavioral problems, and the need for treatment were evaluated around the time of registration to the waiting list and during follow-up. Saitama Medical University Hospital has been designated a hospital for heart transplantation since October 2002. The subjects were 14 heart transplant candidates (9 male candidates and 5 female candidates, mean age 29 years) at the hospital from September 1997 to October 2005. These 14 candidates were equipped with LVADs. The waiting periods on LVAD support were from 119 days to 1028 days, and the average waiting period was 313 days. Six candidates among the 14 had more than one DSM-IV diagnosis. Seven candidates were diagnosed with adjustment disorder, which was the most frequent diagnosis. Three candidates had depressive disorder, one had psychotic disorder, and one had dissociative disorder. Three candidates had acute cognitive dysfunction (delirium) due to their general medical condition. All three had other disorders with mainly psychological elements. Nine candidates (64%) were diagnosed with disorders with mainly psychological elements. Antipsychotics were used for the candidates in psychotic states and with delirium, and there was a need for crisis intervention. Antidepressants and antianxiety drugs were used for the candidates with depressive disorder; they needed intensive observation. Four candidates (28%) needed some attention and some antianxiety drugs or hypnotics. Psychiatric interventions were not necessary in five candidates (36%).

Dopamine D2 receptor-mediated G protein activation assessed by agonist-stimulated [35S]guanosine 5'-O-(gamma-thiotriphosphate) binding in rat striatal membranes.

In order to investigate the functional interaction between the native dopamine receptors and their coupled guanine nucleotide-binding regulatory (G) proteins, dopamine-stimulated [(35)S]guanosine 5'-O-(gamma-thiotriphosphate) ([(35)S]GTPgammaS) binding was pharmacologically characterized in rat striatal membranes. Following optimizing the experimental conditions as to the concentrations of GDP, MgCl(2) and NaCl in the assay medium, the agonist and antagonist properties for a series of dopamine receptor ligands were determined mainly under the standard assay condition. The pharmacological profile of this response clearly indicated the involvement of dopamine D(2)-like receptors, but not of dopamine D(1)-like receptors. Among the types of dopamine D(2)-like receptors, dopamine D(2) receptors most likely appeared to be involved in dopamine-stimulated [(35)S]GTPgammaS binding in rat striatal membranes, because the affinities of agonists and antagonists determined in the present study were significantly correlated with those reported in the previous literature only for dopamine D(2) receptors, but not for dopamine D(3) or D(4) types. Though the concentration-dependent inhibition curves of dopamine-stimulated [(35)S]GTPgammaS binding by spiperone and S(-)-raclopride were apparently biphasic, the origin of the low-affinity minor components was not fully determined. The antiparkinsonian drugs with the properties of dopamine receptor agonism were shown to behave as stimulants with varied affinities and relative efficacies in the current assay system. On the other hand, neither phencyclidine (PCP) nor ketamine stimulated the specific [(35)S]GTPgammaS binding, in contrast with the previous report demonstrating that these two N-methyl-D-aspartic acid (NMDA) receptor antagonists behaved as agonists at human dopamine D(2) receptors expressed in Chinese hamster ovary (CHO) cells. These results provide important information about the functional activation of G proteins coupled with dopamine D(2) receptors as well as agonist actions of various compounds at native dopamine D(2) receptors, which are potentially involved in pathophysiology and pharmacotherapy of neuropsychiatric diseases such as Parkinson's disease, schizophrenia and depression.

5-HT-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes.

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through Gi type G proteins. In the present study, 5-HT-stimulated [35S]GTPgammaS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 microM GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [35S]GTPgammaS binding was stimulated by several compounds that had been shown to be agonists at 5-HT(1B/1D) receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC50) for these agonists were significantly correlated with their pKi's reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [35S]GTPgammaS binding in response to 1 microM 5-HT was potently inhibited by several 5-HT(1B/1D) receptor antagonists as well as beta-adrenoceptor antagonists such as S(-)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 microM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(-)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [35S]GTPgammaS binding was reduced by 1'-methyl-5-([2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4'-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(-)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [35S]GTPgammaS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.

5-HT1A receptor-mediated G protein activation assessed by [35S]GTPgammaS binding in rat cerebral cortex.

To date, 5-hydroxytryptamine1A (5-HT1A) receptor-mediated functional assays (adenylyl cyclase inhibition, high-affinity GTPase activity and [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding) have been performed mainly in hippocampal membranes. In the current study, 5-HT-stimulated G protein activation assays were carried out in rat cerebral cortical membranes. High-affinity GTPase activity was stimulated by 5-HT, but not by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). By contrast, 5-HT- and 8-OH-DPAT-stimulated [35S]GTPgammaS binding displayed sufficient dynamic range enough to warrant further pharmacological analysis. Under standard conditions, which were determined precisely in terms of the concentrations of GDP, MgCl2 and NaCl, the profile of 5-HT-stimulated [35S]GTPgammaS binding investigated using a series of 5-HT receptor agonists and antagonists clearly indicated the involvement of the 5-HT1A receptor subtype. There appeared to be no evidence supporting the presence of regional heterogeneity in coupling efficiency between 5-HT1A and G proteins in the hippocampus or cortex. This method is a useful tool for investigating functional coupling between postsynaptic 5-HT1A receptors and G proteins in cerebral cortical membranes.

Trazodone and its active metabolite m-chlorophenylpiperazine as partial agonists at 5-HT1A receptors assessed by [35S]GTPgammaS binding.

Trazodone is an effective antidepressant drug with a broad therapeutic spectrum, including anxiolytic efficacy. Although trazodone is usually referred to as a serotonin (5-HT) reuptake inhibitor, this pharmacological effect appears to be too weak to fully account for its clinical effectiveness. The present study aimed to elucidate the agonist properties of trazodone and its active metabolite, m-chlorophenylpiperazine (m-CPP), at 5-HT(1A) receptors by means of the guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding assay. In membranes prepared from Chinese hamster ovary cells expressing human 5-HT(1A) receptors (CHO/h5-HT(1A)), trazodone behaved as an almost full agonist and m-CPP was also a highly efficacious partial agonist at 5-HT(1A) receptors. The intrinsic activities of both compounds were higher than those of tandospirone and buspirone, which are clinically effective anxiolytics with well-known 5-HT(1A) partial agonist properties. These effects were replicated in the 5-HT(1A) receptor-mediated [(35)S]GTPgamma(S) binding assay in native rat brain membranes (at least in hippocampal membranes), although the intrinsic activities of the compounds were low and differently ranked compared to those in CHO/h5-HT(1A) cell membranes. When considering the implications of 5-HT(1A) receptors in anxiety and/or depression, as well as the clinical effectiveness of azapirone anxiolytics with partial 5-HT(1A) receptor agonist properties such as buspirone, it is possible that the agonist effects on 5-HT(1A) receptors of trazodone and its active metabolite m-CPP presented in this study contribute, at least in part, to the clinical efficacy of the atypical antidepressant trazodone.

Detailed pharmacological characterization of 5-HT1A-receptor-mediated [35S]GTP gamma S binding in rat hippocampal membranes.

5-HT-stimulated [(35)S]GTPgammaS binding to rat hippocampal membranes was pharmacologically characterized. Signal/noise ratio or percent increase over basal was optimized with 100 microM GDP, 2-10 mM MgCl(2), and 150-200 mM NaCl. However, we preferred the standard condition (20 microM GDP, 5 mM MgCl(2), and 100 mM NaCl: Condition I) to the alternative one (100 microM GDP, 5 mM MgCl(2), and 150 mM NaCl: Condition II) because 1) absolute values of basal and 5-HT-sensitive bindings decreased with higher concentrations of GDP and NaCl; 2) EC(50) values determined under Condition II were 2 - 6 fold higher than those under Condition I; 3) some partial agonists had less intrinsic activities in the presence of higher concentrations of GDP; and 4) Inhibitory effects of WAY100635 were complete under Condition I, while incomplete under Condition II. Pharmacological profile of concentration-dependent stimulation by a series of 5-HT ligands and concentration-dependent inhibition of 5-HT-stimulated binding by several 5-HT-receptor antagonists clearly indicated that this response under Condition I was mediated solely through 5-HT(1A) receptors. Although caution should be paid especially to the apparent intrinsic activities susceptible to the assay conditions, this method appears useful to investigate functional coupling between 5-HT(1A) receptors and their coupled G proteins in native hippocampal membranes.