GTPgammaS Binding Research Articles

Measurement of agonist-stimulated [35S]GTPgammaS binding to assess total G-protein and Galpha-subtype-specific activation by G-protein-coupled receptors.

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through promotion of guanine nucleotide exchange on the Galpha-subunit of heterotrimeric G-proteins to release free Galpha-GTP and betagamma-subunits. In membrane preparations, GTP can be substituted by 35S-labeled guanosine 5'-O-(3-thio)triphosphate ([35S]GTPgammaS) and on agonist stimulation a stable [35S]GTPgammaS-Galpha complex will form and accumulate. Separation of 35S-bound GTPgammaS-Galpha complexes from free [35S]GTPgammaS allows differences between basal and agonist-stimulated rates of [35S]GTPgammaS-Galpha complex formation to be used to obtain pharmacological information on receptor-G-protein information transfer. Further, by releasing Galpha-subunits into solution following the [35S]GTPgammaS binding step, Galpha-subunit-specific antibodies can be used to investigate the Galpha-protein subpopulations activated by receptors by immunoprecipitation of [35S]GTPgammaS-Galpha complexes and quantification by scintillation counting. Here we describe a total [35S]GTPgammaS binding assay and a modification of this method that incorporates a Galpha-specific immunoprecipitation step.

New endomorphin analogs with mu-agonist and delta-antagonist properties

Abstract Endomorphins (endomorphin-1,H-Tyr-Pro-Trp-Phe-NH 2 ,endomorphin-2, Tyr-Pro-Phe-Phe-NH2) are potent and selective µ-opioid receptor agonists. In order to improve the affinity and chemical stability of endomorphins, we have designed, synthesized, and characterized novel analogs with unnatural (2 ',6 '-dimethyltyrosine, Dmt) and/or ß-alicyclic amino acids (ACPC and ACHC). Radioligand binding assay indicated that several of the novel analogs exhibit high affinity for both µ-and d-opioid receptors in rat-or mouse-brain membrane preparations. The most promising derivatives—such as Dmt-Pro-Trp/Phe-Phe-NH2, Dmt-(1 S,2R)-ACPC-Phe-Phe-NH2, and Dmt-(1S,2R)-ACHC-Phe-Phe-NH2 )—were characterized in recombinant cell lines expressing human µ-or d-opioid receptors. Interestingly, while these novel peptides were potent opioid agonists in the functional [35S]GTPgammaS binding assays in Chinese hamster ovary cells expressing the µ-opioid receptors, some behaved as antagonist or inverse agonist in the human d-opioid receptor-expressing CHO cells. Since it has previously been demonstrated that the coadministration of d-antagonists with µ-analgesics attenuates the development of analgesic tolerance, introduction of high-affinity d-antagonist properties into the µ-agonist endomorphins is expected to lead to potent analgesics that produce limited tolerance.

Purification and In Vitro Functional Analyses of RGS12 and RGS14 GoLoco Motif Peptides

The GoLoco motif is a short polypeptide sequence that binds to heterotrimeric G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) subclass in a nucleotide-dependent manner (i.e., solely to the GDP-bound ground state). This article describes methods used for the expression, purification, and in vitro evaluation of membrane-permeant tag fusion peptides derived from the GoLoco motif regions of "regulator of G-protein signaling" proteins type 12 (RGS12) and 14 (RGS14) and a consensus GoLoco sequence from the multiple GoLoco motif protein AGS3. Three different fluorescence-based assays are described for evaluating the in vitro function of these GoLoco peptides as guanine nucleotide dissociation inhibitors, including measurements of GTPgammaS binding and Galpha subunit activation by the planar ion aluminum tetrafluoride.

Structure-activity study on the Phe side chain arrangement of endomorphins using conformationally constrained analogues.

Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residues to generate a conformationally constrained peptide set. This series of molecules was subjected to biological assays, and for beta-MePhe(4)-endomorphins-2, a conformational analysis was performed. Incorporation of (2S,3S)-beta-MePhe(4) resulted in the most potent analogues of both endomorphins with enhanced enzymatic stability. Their micro opioid affinities were 4-times higher than the parent peptides, they stimulated [(35)S]GTPgammaS binding, and they were found to be full agonists. NMR experiments revealed that C-terminal (2S,3S)-beta-MePhe in endomorphin-2 strongly favored the gauche (-) spatial orientation which implies the presence of the chi(1) = -60 degrees rotamer of Phe(4) in the binding conformer of endomorphins. Our results emphasize that the appropriate orientation of the C-terminal aromatic side chain of endomorphins is substantial for binding to the micro opioid receptor.

Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPgammaS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.

Identification of Drosophila Neuropeptide Receptors by G Protein-coupled Receptors-β-Arrestin2 Interactions

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract Guanine nucleotide effects on agonist binding.

Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 microM) and KD-low ( approximately 10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 microM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.

Direct evidence for the up-regulation of spinal micro-opioid receptor function after repeated stimulation of kappa-opioid receptors in the mouse.

The present study was designed to investigate the possible change in spinal micro -opioid receptor function after repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] in the ICR mouse. A single s.c. or i.t. injection of (-)U-50,488H produced a dose-dependent antinociception. Repeated s.c. or i.t. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect induced by the i.t. administration of a selective micro -opioid receptor agonist [d-Ala2,N-Me-Phe4,Gly5-ol] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTPgammaS) binding assay, we found that (-)U-50,488H was able to produce a dose-dependent increase in [35S]GTPgammaS binding to membranes of the mouse spinal cord. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTPgammaS binding in this region, whereas repeated treatment with (-)U-50,488H exhibited an increase in the DAMGO-stimulated [35S]GTPgammaS binding in membranes of the spinal cord. Using a receptor binding assay, repeated treatment with (-)U-50,488H significantly increased the density of [3H]DAMGO binding sites in membranes of the mouse spinal cord. In contrast, the expression of micro -opioid receptor was not affected after repeated treatment with (-)U-50,488H. These results suggest that repeated stimulation of kappa-opioid receptors leads to the up-regulation of micro -opioid receptor functions in the spinal cord, which may be associated with an increase in the number of functional micro -opioid receptors in the mouse spinal cord.

The rabbit motilin receptor: molecular characterisation and pharmacology.

Following identification of the human motilin receptor, we isolated the rabbit orthologue by PCR amplification and found this to be 85% identical to the open reading frame of the human receptor. The protein encoded was 84% identical to the human polypeptide. In HEK293T cells transfected with the rabbit receptor, motilin concentration-dependently increased intracellular calcium mobilisation (pEC50=9.25). After transfection with Go1alpha, motilin similarly stimulated [35S]GTPgammaS binding (pEC50=8.87). Using both systems, similar values were obtained with the human receptor, with rank-order potencies of motilin=[Nle13]-motilin>erythromycin; ghrelin was ineffective. In circular muscle preparations of rabbit gastric antrum, [Nle13]-motilin 0.1-30 nM concentration-dependently increased the amplitude of electrically-evoked, neuronally-mediated contractions (pEC50=8.3); higher concentrations increased the muscle tension (30-3000 nM). Both responses to [Nle13]-motilin faded rapidly during its continual presence. Rat or human ghrelin 0.01-10 microM were without activity. Erythromycin 30-3000 nM and 10 microM, respectively, increased neuronal activity and muscle tension in rabbit stomach. Unlike [Nle13]-motilin, the increase in neuronal activity did not fade during continual presence of submaximally-effective concentrations of erythromycin; some fade was observed at higher concentrations. We conclude that the pharmacology of the rabbit motilin receptor is similar to the human orthologue and, when expressed as a recombinant, comparable to the native receptor. However, in terms of their ability to increase neuronal activity in rabbit stomach, [Nle13]-motilin and erythromycin are distinguished by different response kinetics, reflecting different rates of ligand degradation and/or interaction with the receptor.

Design and synthesis of novel dimeric morphinan ligands for kappa and micro opioid receptors.

A novel series of morphinans were synthesized, and their binding affinity at and functional selectivity for micro, delta, and kappa opioid receptors were evaluated. These dimeric ligands can be viewed as dimeric morphinans, which were formed by coupling two identical morphinan pharmacophores (cyclorphan (1) or MCL 101 (2)) with varying connecting spacers. Ligands 6 and 7 with alkyl spacers on the nitrogen position and ligands 8 and 9 in which the two morphinan pharmacophores were coupled by ether moieties at the 3-hydroxyl positions showed significant decrease in affinity at all three opioid receptors. An improvement in the affinity was achieved by introducing an ester moiety as the spacer in the dimeric morphinans. It was observed that the affinity of these ligands was sensitive to the character and length of the spacer. Compound 13 (MCL-139) with a 4-carbon ester spacer, compound 17 (MCL-144) containing a 10-carbon spacer, and compound 19 (MCL-145) with the conformationally constrained fumaryl spacer were the most potent ligands in this series, displaying excellent affinities at micro and kappa receptors (K(i) = 0.09-0.2 nM at micro and K(i) = 0.078-0.049 nM at kappa), which were comparable to the parent compound 2. Ligand 12, a compound containing only one morphinan pharmacophore and a long-chain ester group, had affinity at both micro and kappa receptors almost identical to that of the parent ligand 2. In the [(35)S]GTPgammaS binding assay, ligands 13, 17, and 19 and their parent morphinans 1 and 2 stimulated [(35)S]GTPgammaS binding mediated by the micro and kappa receptors. Compounds 13 and 17 were full kappa agonists and partial micro agonists, while compound 19 was a partial agonist at both micro and kappa receptors. These novel ligands, as well as their interesting pharmacological properties, will serve as the basis for our continuing investigation of the dimeric ligands as potential probes for the pharmacotherapy of cocaine abuse and may also open new avenues for the characterization of opioid receptors.

Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm.

A series of mu opioid receptor gene Oprm splice variants have been reported that differ only at their C-terminus. These variants all contain exons 1, 2, and 3 of the gene, the exons responsible for coding all seven transmembrane domains. Whereas MOR-1 also has exon 4 that encodes for an additional 12 amino acids at the tip of the C-terminus, the other MOR-1 variants have unique amino acid sequences distinct from those in MOR-1 due to alternative splicing. All these variants are mu-selective in binding assays. The current study explored the ability of these variants to stimulate [35S]GTPgammaS binding to assess them functionally. Only mu opioids stimulated [35S]GTPgammaS binding. Among the mu opioids we noted marked differences in their maximal stimulation among the clones. This was most prominent with beta-endorphin, which stimulated [35S]GTPgammaS binding in the MOR-1E expressing cells to a greater degree than [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 130%) and was far less effective than DAMGO in MOR-1C cells (44%). The rank order of maximal stimulation of the drugs varied among the clones as well. Dynorphin A, beta-endorphin and morphine were most effective in stimulating [35S]GTPgammaS binding in MOR-1E, while M6G and fentanyl were most effective in MOR-1 expressing cells. The potency (EC50) of some of the drugs also varied extensively among the clones, with a poor correlation between the potency of the drugs to stimulate [35S]GTPgammaS binding and their binding affinity. Together, these findings reveal marked functional differences among the variants that only can be explained by their structural differences at the tip of their C-terminus.

Functional Annotation of the Putative Orphan Caenorhabditis elegans G-protein-coupled Receptor C10C6.2 as a FLP15 Peptide Receptor

This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [35S]guanosine 5'-O-(thiotriphosphate) (GTPgammaS) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B >> F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTPgammaS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 degrees C. However, a 37 to 28 degrees C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

The histamine H1 receptor and the alpha1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([35S]thio)triphosphate ([35S]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [35S]GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner.

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.

Pharmacological Characterization and Identification of Amino Acids Involved in the Positive Modulation of Metabotropic Glutamate Receptor Subtype 2

In the present study, we describe the characterization of a positive allosteric modulator at metabotropic glutamate subtype 2 receptors (mGluR2). N-(4-(2-Methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379) is a selective positive allosteric modulator at human mGluR2 and is without activity at human mGluR3. Furthermore, LY487379 has no intrinsic agonist or antagonist activity at hmGluR2, as determined by functional guanosine 5'(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding, single-cell Ca2+ imaging, and electrophysiological studies. However, LY487379 markedly potentiated glutamate-stimulated [35S]GTPgammaS binding in a concentration-dependent manner at hmGluR2, shifting the glutamate dose-response curve leftward by 3-fold and increasing the maximum levels of [35S]GTPgammaS stimulation. This effect of LY487479 was also observed to a greater extent on the concentration-response curves to selective hmGluR2/3 agonists. In radioligand binding studies to rat cortical membranes, LY487379 increased the affinity of the radiolabeled agonist, [3H]DCG-IV, without affecting the binding affinity of the radiolabeled antagonist, [3H]LY341495. In rat hippocampal slices, coapplication of LY487379 potentiated synaptically evoked mGluR2 responses. Finally, to elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR2 and hmGluR3. Substitution of Ser688 and/or Gly689 in transmembrane IV along with Asn735 located in transmembrane segment V, with the homologous amino acids of hmGluR3, completely eliminated LY487379 allosteric modulation of hmGluR2. We propose that this allosteric binding site defines a pocket that is different from the orthosteric site located in the amino terminal domain.

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase, phosphatidylinositol 3-kinase, Akt and mitogen-activated protein kinase.

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.

Ca2+ responses in Chinese hamster ovary-K1 cells demonstrate an atypical pattern of ligand-induced 5-HT1A receptor activation.

Little experimental evidence has been reported for diverse signaling via 5-hydroxytryptamine (5-HT)1A receptors despite the fact that agonists seem to be more efficacious at dorsal raphe somatodendritic 5-HT1A autoreceptors than at postsynaptic 5-HT1A receptors. The present study investigated Ca2+ responses in Chinese hamster ovary (CHO)-K1 cells expressing a human 5-HT1A receptor by 5-HT, prototypical 5-HT1A agonists, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methyl-6-; methylaminopyridin-2-yl)-methylaminomethyl]-piperidine (F 14679), and especially N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-; methylaminomethyl]piperidine (F 13640) as representative ligands of a new chemical class (methylamino-pyridine) that combines both high efficacy and selectivity for 5-HT1A receptors. 5-HT (pEC50 = 6.70 +/- 0.02) induced a pertussis toxin-sensitive, transient high-magnitude Ca2+ response. High-magnitude Ca2+ responses (Emax, percentage versus 5-HT) were also found with F 13640 (107 +/- 4), 5-carboxamidotryptamine (100 +/- 3), and F 14679 (87 +/- 3). In contrast, the prototypical 5-HT1A receptor agonists buspirone, ipsapirone, and 8-(hydroxy-2-(di-n-propylamino)tetralin, and also flesinoxan and eptapirone, were virtually inactive (< or =5). This atypical pattern of 5-HT1A receptor activation contrasts with the broad spectrum of the ligands' partial agonist properties as observed by measuring guanosine 5'-O-(3-[35 S]thio)triphosphate ([35S]GTPgammaS) binding responses with membranes of either CHO-K1 or C6-glial cells stably expressing a human 5-HT1A receptor. Remarkably, differences between ligands that seem small in the [35S]GTPgammaS binding assay translate into huge differences in the magnitude of Ca2+ responses. Therefore, some of these 5-HT1A ligands (i.e., F 13640) may in a selective way induce responses that may be not at all be achieved with other ligands (i.e., buspirone). In conclusion, the pharmacology of 5-HT1A receptor ligands seems to be codetermined by the effector pathway.

Hyperactivity to novelty induced by social isolation is not correlated with changes in D2 receptor function and binding in striatum.

Prolonged social isolation has been reported to induce different behavioral disturbances, among the most consistent of which are the increased locomotor response to novelty and the effects of psychostimulants. While these behavioral changes have been partly related to a dysregulation of dopaminergic activity in striatum (dorsal and ventral), the involvement of changes in the function of dopamine receptors is still a matter of controversy. To investigate the effects of prolonged social isolation on the function of D2 receptors at both the behavioral and biochemical levels. Sprague-Dawley rats were randomly placed at 21 days of age in groups or isolation for 2 months. Horizontal and vertical locomotor activities induced by novelty and also by systemic injections of the D2 agonist quinpirole (0.15, 0.50 and 1.5 mg/kg i.p.) and their modulation by the A2A agonist CGS 21680 (0.1 mg/kg i.p.) were studied. The effects of social isolation on the avoidance learning assessed by the passive avoidance test were also studied. Binding experiments were performed to study the number and affinity of D2 receptors by means of saturation and competition experiments with the D2 antagonist [(3)H]-raclopride and the interaction between D2 receptors and the G-protein by means of [(35)S]-GTPgammas binding in dorsal/ventral striatal membranes of both grouped and isolated rats. Rats reared in isolation were hyperactive to a novel environment and showed shorter retention latencies in the passive avoidance test. Isolation rearing did not modify the increase in motor activity produced by quinpirole nor the counteraction of these effects by the simultaneous stimulation of A2A receptors. Likewise, the number, affinity and functional efficacy of D2 receptors were not changed by social isolation. These results suggest that the hyperactivity to novelty and psychostimulants as well as other behavioral changes induced by social isolation do not parallel changes in the in vivo function or binding of D2 receptors in dorsal/ventral striatum.

Up-regulation of spinal muscarinic receptors and increased antinociceptive effect of intrathecal muscarine in diabetic rats.

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors produce effective pain relief. Intrathecal injection of a small dose of neostigmine produces a profound antiallodynic effect in rats with diabetic neuropathy. However, the mechanisms of increased antinociceptive effect of cholinergic agents on diabetic neuropathic pain are not clear. In the present study, we tested the hypothesis that spinal muscarinic receptors are up-regulated in diabetes. The withdrawal threshold of the hindpaw in response to noxious heat and pressure stimuli was determined in streptozotocin-induced diabetic and age-matched normal rats. Muscarine-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding was used to assess the change of functional muscarinic receptors in the spinal cord in diabetes. The [3H]AF-DX 384 membrane binding was performed to determine the number and affinity of spinal cord M2 muscarinic receptors in normal and diabetic rats. We found that the antinociceptive effect of intrathecal 2 to 12 mug muscarine in diabetic animals was potentiated significantly compared with that in normal animals. The maximal muscarine-stimulated [35S]GTPgammaS binding was 112.5 +/- 8.3% in normal rats and 168.8 +/- 12.1% (P < 0.05) in diabetic rats. Although the KD value (2.9 nM) was similar in both groups, the Bmax of [3H]AF-DX 384 membrane binding was significantly higher in diabetic than in normal rats (255.2 +/- 5.9 versus 165.9 +/- 3.5 fmol/mg protein, P < 0.05). Collectively, these data strongly suggest that the muscarinic receptor is up-regulated in the dorsal spinal cord in diabetic rats. This finding probably accounts for the increased efficacy of the antinociceptive effect of intrathecal muscarinic agonists in diabetic neuropathic pain.

N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds: novel allosteric enhancers of gamma-aminobutyric acidB receptor function.

N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABA(B) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes from a GABA(B)(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [35S]GTPgammaS binding by themselves. Similar effects of GS39783 are seen on native GABA(B) receptors in rat brain membranes. Concentration-response curves with GABA in the presence of different fixed concentrations of GS39783 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B)(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [3H]3-aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [3H]CGP62349), GS39783 increases agonist affinities. Agonist displacement curves are biphasic, probably reflecting the G protein-coupled and uncoupled states of the receptor. The proportion of the high-affinity component is increased by GS39783, suggesting that the G protein coupling of the receptor is also promoted by the positive modulator. We also show that GS39783 has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca2+ signaling in human embryonic kidney 293 cells. In a more physiological context, GS39783 is shown to suppress paired pulse inhibition in rat hippocampal slices. This effect is reversed by the competitive GABA(B) receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABA(B) receptors.