Agonist-stimulated [35S]GTPgammaS binding in brain modulation by endogenous adenosine.
Coupling of receptors to G-proteins can be assessed by the ability of specific agonists to stimulate [35S]GTPgammaS 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]GTPgammaS binding. The present study suggests a modification of the assay to reduce basal [35S]GTPgammaS binding and thus increase the signal:noise ratio. Adenosine A1 receptors belong to the class of G-protein-coupled receptors that activate Gi/Go proteins in brain. In the present study, the A1 agonist R(-)N6-(2-phenylisopropyl)adenosine (R-PIA) stimulated [35S]GTPgammaS binding in brain regions known to contain A1 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]GTPgammaS binding. In addition, for cannabinoid and mu-opioid agonists, the percent stimulation of [35S]GTPgammaS binding was approximately doubled when ADase was included in the assay. These results suggest that endogenous adenosine contributes significantly to basal [35S]GTPgammaS 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]GTPgammaS binding.
Moore
R J
RJ
Department of Physiology and Pharmacology and Center for Investigative Neuroscience, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Xiao
R
R
Sim-Selley
L J
LJ
Childers
S R
SR
D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D008297:Male; D058906:Purinergic P1 Receptor Agonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D013462:Sulfur Radioisotopes
D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000243:Adenosine Deaminase; D019204:GTP-Binding Proteins; D000241:Adenosine