AUTORADIOGRAPHIC CHARACTERIZATION OF [H-3] CGMP BINDING-SITES IN THE RAT-BRAIN

Quantitative autoradiography was used to characterize and localize [H- 3]cGMP binding sites in the rat brain. [H-3]cGMP binding was found to be pH-sensitive (with two optima at 7.4 and 5.0) and Mg2(+)-dependent. At pH 7.4, the binding was dependent on inclusion of the phosphodiest erase inhibitor IBMX. In contrast, at pH 5.0, IBMX had little effect o n binding. The binding of [H-3]cGMP was reversible and saturable with a K-d of 22 nM at pH 7.4 and 36 nM at pH 5.0. B-max values were 172 fm ol/mg at pH 7.4 and 462 fmol/mg at pH 5.0. [H-3]cGMP binding was inhib ited by cGMP and its analogues, with cGMP and cAMP being the most pote nt at pH 7.4 and cGMP and 8-Br-cGMP being the most potent at pH 5.0. U sing an extracellular pH 7.4 buffer, the selective cGMP-dependent prot ein kinase (PKG) inhibitor Rp-8pCPT-cGMPS had very little effect on [H -3]cGMP binding. In contrast, with a cytosolic pH 5.0 buffer, Rp-8pCPT -cGMPS displaced binding in the cerebellum. This indicates that PKG is localized in the cerebellum, and that the binding to PKG is favored u nder cytosolic conditions. Autoradiographic localization of [H-3]cGMP binding sites revealed a heterogeneous distribution with the highest d ensities in the substantia nigra and interpeduncular nucleus. High den sities were also observed in the basal ganglia, the medial habenular n ucleus, the frontoparietal cortex, the lateral amygdaloid nucleus and the subiculum. It is concluded that the nature of [H-3]cCMP binding is complex, with one site probably being related to cytosolic PKG mainly found in the cerebellum, and one site probably representing cGMP-stim ulated phosphodiesterase mainly located in the forebrain. (C) 1997 Els evier Science B.V.