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 View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2006-02

AUTHORS

Yuji Odagaki, Ryoichi Toyoshima

ABSTRACT

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. More... »

PAGES

335-345

References to SciGraph publications

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s00210-006-0041-x

    DOI

    http://dx.doi.org/10.1007/s00210-006-0041-x

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1018542902

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/16491386


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