Agonist-stimulated [S-35]GTP gamma S autoradiography: optimization for high sensitivity

The receptor-stimulated accumulation of [S-35]GTP gammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autor adiographic visualization of [S-35]GTP gammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specifi c (NSB). basal and agonist-stimulated [S-35]GTP gammaS binding were measure d, using methadone, 5-carboxamidotryptamine and epinephrine for mu -opiate receptors, 5-HT1A receptors and alpha (2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist -stimulated [S-35]GTP gammaS binding was more easily observed. Further opti mization using glycylglycine buffer found increased signal-to-noise ratio w ith inclusion of dithiothrietol, increased [S-35]GTP gammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [S-35]GTP gammaS at 0.1 nM. Improved sensitivity was due to decrease d NSB and basal [S-35]GTP gammaS binding and agonist-stimulated binding wer e similarly affected for each receptor system. The assay conditions describ ed should extend the use of agonist-stimulated [S-35]GTP gammaS autoradiogr aphy to receptors, which produce low levels of [S-35]GTP gammaS binding and to the measurement of changes in receptor-G protein coupling. (C) 2001 Pub lished by Elsevier Science B.V.