IN-VITRO AUTORADIOGRAPHY OF RECEPTOR-ACTIVATED G-PROTEINS IN RAT-BRAIN BY AGONIST-STIMULATED GUANYLYL 5'-[GAMMA-[S-35]THIO]TRIPHOSPHATE BINDING

Agonists stimulate guanylyl 5'-[gamma-[S-35]thio]-triphosphate (GTP[ga mma-S-35]) binding to receptor-coupled guanine nucleotide binding prot ein (G proteins) in cell membranes as revealed in the presence of exce ss GDP, We now report that this reaction can be used to neuroanatomica lly localize receptor-activated G proteins in brain sections by in vit ro autoradiography of GTP[gamma-S-35] binding. Using the mu opioid-sel ective peptide [D-Ala(2),N-MePhe(4),Gly(5)-ol] enkephalin (DAMGO) as a n agonist in rat brain sections and isolated thalamic membranes, agoni st stimulation of GTP[gamma-S-35] binding required the presence of exc ess GDP (1-2 mM GDP in sections vs, 10-30 mu M GDP in membranes) to de crease basal G-protein activity and reveal agonist-stimulated GTP[gamm a-S-35] binding. Similar concentrations of DAMGO were required to stim ulate GTP[gamma-S-35] binding in sections and membranes, To demonstrat e the general applicability of the technique, agonist-stimulated GTP[g amma-S-35] binding in tissue sections was assessed with agonists for t he mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyri c acid type B (baclofen) receptors, For opioid and cannabinoid recepto rs, agonist stimulation of GTP[gamma-S-35] binding was blocked by incu bation with agonists in the presence of the appropriate antagonists (n aloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrat ing that the effect was specifically receptor mediated, The anatomical distribution of agonist-stimulated GTP[gamma-S-35] binding qualitativ ely paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that varia tions in coupling efficiency may exist between different receptors in various brain regions, This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins b y specific receptors.