Characterization of receptor-mediated [35S]GTPgammaS binding to cortical membranes from postmortem human brain.
The [35S]GTPgammaS binding assay represents a functional approach to assess the coupling between receptors and G-proteins. The optimal conditions for [35S]GTPgammaS binding to human brain homogenates were established in postmortem samples of prefrontal cortex. The influence of protein content, incubation time, GDP, Mg(2+), and NaCl concentrations on the [35S]GTPgammaS binding were assessed in the absence and presence of the alpha(2)-adrenoceptor agonist UK14304 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine). In conditions of 50 microM GDP and 100 mM NaCl, UK14304 increased the apparent affinity of the specific [35S]GTPgammaS binding without changing the apparent density. Concentration-response curves to agonists of alpha(2)-adrenoceptors, mu-opioid, 5-HT(1A), cholinergic muscarinic, and GABA(B) receptors displayed, in the presence of NaCl, maximal stimulations between 24% and 61% with EC(50) values in the micromolar range. Selective antagonists shifted to the right the agonist-induced stimulation curves. The G(i)/G(o)-protein alkylating agent N-ethylmaleimide decreased basal [35S]GTPgammaS binding in a concentration-dependent manner and inhibited the stimulation induced by the different agonists. In cortical sections, [35S]GTPgammaS binding to gray matter was stimulated by the agonist UK14304. The present study demonstrates that functional studies of the receptor coupling to G(i)/G(o)-proteins can be performed in postmortem human brain samples.
González-Maeso
J
J
Departamento de Farmacología, Universidad del País Vasco/Euskal Herriko Unibertsitatea, E-48940 Leioa, Bizkaia, Spain.
Rodríguez-Puertas
R
R
Gabilondo
A M
AM
Meana
J J
JJ
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D001345:Autoradiography; D002540:Cerebral Cortex / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013268:Stimulation, Chemical; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013997:Time Factors
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D013439:Sulfhydryl Reagents; D013462:Sulfur Radioisotopes; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide