CYCLIC GMP-DEPENDENT PROTEIN-KINASE ACTIVATION MEDIATES INHIBITION OFCATECHOLAMINES SECRETION AND CA2+ INFLUX IN BOVINE CHROMAFFIN CELLS

The effects of the membrane-permeable cGMP analogue, 8-bromoguanosine 3':5'-cyclic monophosphate on acetylcholine-evoked catecholamine secre tion and cytosolic calcium increases were studied in chromaffin cells from the bovine adrenal gland. Preincubation with 100 mu M 8-bromoguan osine 3':5'-cyclic monophosphate during 10 and 30 min decreased the ac etylcholine-evoked catecholamine release by 16 +/- 3% and 27 +/- 5%, r espectively. The cytosolic calcium increases triggered by acetylcholin e and 30 mM KCl were also inhibited by 30 min of preincubation with th is compound by 27 +/- 4 and 34 +/- 2%, respectively. Changes in membra ne potential induced by acetylcholine and KCl were not affected by pre incubation with 8-bromoguanosine 3':5'-cyclic monophosphate. The cycli c GMP-dependent protein kinase inhibitor N-[2-(methylamino)ethyl]-5-is oquinoline sulfonamide dihydrochloride- at 1 mu m abolished the inhibi tory effect of 8-bromoguanosine 3':5'-cyclic monophosphate on acetylch oline-evoked calcium increase. By contrast, a potent and selective inh ibitor against cyclic AMP-dependent protein kinase, romocinnamylamino) ethyl]-5-isoquinolinesulfonamide did not block the 8-bromoguanosine 3' :5'-cyclic monophosphate effect. Additionally, 8-bromoguanosine 3': 5' cyclic monophosphate stimulated histone F-2b phosphorylation by a part ial purified cGMP-dependent protein kinase from chromaffin cells. The extent of histone phosphorylation was reduced by N-[2-(methylamino)eth yl]-5-isoquinolinesulfonamide dihydrochloride and 8-(4-chlorophenylthi o)-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer, a specific inhibitor against cyclic GMP-dependent protein kinase, whereas it was not modified by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfo namide. The results suggest that the inhibitory effects of 8-bromoguan osine 3':5' cyclic monophosphate on chromaffin cells are mediated thro ugh the activation of cGMP-dependent protein kinase. The target for th is kinase does not appear to be either the nicotinic receptor or potas sium channels, but it could be proteins which participate in Ca2+ infl ux or in the maintenance of calcium levels in chromaffin cells.