Hydrolysis‐Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin‐Permeabilized PC12 Cells

Abstract

The effect of the hydrolysis-resistant GTP analogs, guanosine 5''-O-(3-thiotriphosphate) (GTP.gamma.S) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca2+ (10 .mu.M) was not affected by GTP.gamma.S or GMMPPNP, secretion in the absence of Ca2+ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTP.gamma.S or GMPPNP was approximately 80% of that induced by 10 .mu.M Ca2+. Half-maximum stimulation was induced by 30 .mu.M GTP.gamma.S or GMPPNP. Both Ca2+-stimulated and GTP.gamma.S-stimulated secretion were ATP dependent and inhibited by N-ethylmaleimide. The GTP.gamma.S-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca2+ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12-O-tetradecanoylphorbol 13-acetate caused a 50% increase in both Ca2+-stimulated and GTP.gamma.S-stimulated secretion. Cholera and pertussis toxins did not affect Ca2+-stimulated or GTP.gamma.S-stimulated NE secretion. Guanosine 5''-O-(2-thiodiphosphate) (GDP.beta.S) and GTP inhibited GTP.gamma.S-stimulated secretion but not Ca2+-stimulated secretion. The inability of GDP.beta.S to inhibit Ca2+-stimulated secretion indicates that the process affected by GTP.gamma.S is not an essential step in the Ca2+-stimulated pathway.