Agonist-stimulated [ 35S]GTPγS binding in brain : Modulation by endogenous adenosine

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Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [ 35S]GTPγS 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 [ 35S]GTPγS binding. The present study suggests a modification of the assay to reduce basal [ 35S]GTPγS binding and thus increase the signal:noise ratio. Adenosine A 1 receptors belong to the class of G-protein-coupled receptors that activate G i/G o proteins in brain. In the present study, the A 1 agonist R(-)N 6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [ 35S]GTPγS binding in brain regions known to contain A 1 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 [ 35S]GTPyS binding. In addition, for cannabinoid and μ-opioid agonists, the percent stimulation of [ 35S]GTPγS binding was approximately doubled when ADase was included in the assay. These results suggest that endogenous adenosine contributes significantly to basal [ 35S]GTPγS 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 [ 35S]GTPγS binding.