Agonist-stimulated [S-35]GTP gamma S binding in brain - Modulation by endogenous adenosine

Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [S-35]GYP gamma S binding in both brain membranes and sections in the presence of excess GDP, In some brain regions, however, high basal activity makes it difficult to detect agonist-stimulated [S-35]GTP gamma S binding. The present study suggests a modification of the assay to reduce basal [S-35]CTP gamma S binding and thus increase the signal:noise ratio. Adenosine A, receptors belong to the class of G-protein-coupled receptors that activate G(i)/G(0) proteins in brain. In the present study, the A, agonist R(-)N-6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [S-35]GTP gamma S binding in brain regions known to contain A, receptors, including cerebellum, hippocampus and dentate gyrus, medial geniculate body, superior colliculus, certain thalamic nuclei, cerebral cortex, piriform cortex, caudate-putamen, and nucleus accumbens. Treatment of sections and membranes with adenosine deaminase (ADase), which is typically used in adenosine assays to eliminate endogenous adenosine, reduced basal [S-35]GTP gamma S binding. In addition, for cannabinoid and mu-opioid agonists, the percent stimulation of [S-35]GTP gamma S binding was approximately doubled when ADase was included in the assay, These results suggest that endogenous adenosine contributes significantly to basal [S-35]GTP gamma S binding in certain brain regions, and that this activity may be reduced by the addition of ADase, thus improving the signal:noise:ratio of agonist-stimulated [S-35]GTP gamma S binding. (C) 2000 Elsevier Science Ltd. All rights reserved.