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

The receptor-stimulated accumulation of [35S]GTPgammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [35S]GTPgammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [35S]GTPgammaS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for mu-opiate receptors, 5-HT(1A) receptors and alpha(2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [35S]GTPgammaS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [35S]GTPgammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [35S]GTPgammaS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [35S]GTPgammaS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [35S]GTPgammaS autoradiography to receptors, which produce low levels of [35S]GTPgammaS binding and to the measurement of changes in receptor-G protein coupling.