Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is mimicked by 8-bromo cyclic GMP.

Abstract

The effects of sodium nitroprusside, 8-bromo cyclic GMP, 8-bromoguanosine 5'-monophosphate, 8-bromo cyclic AMP, dibutyryl cyclic AMP, and isoproterenol on incorporation of (32)P into proteins in intact rat thoracic aorta were studied. Aortas were incubated in [(32)P]orthophosphate in order to label endogenous adenosine triphosphate. Agents were then added for various times and the tissues were homogenized and fractionated (100,000 x g for 60 min) into soluble and particulate fractions. Soluble and particulate fractions were subjected to isoelectric focusing followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiographs were made. Nitroprusside induced a concentration-dependent increase in incorporation of (32)P into nine proteins and a decrease in (32)P incorporation into two proteins. Some of these proteins appeared in both the soluble and particulate fractions of homogenates; others appeared only in the soluble fraction. The pattern of (32)P incorporation was identical after 2- or 15-min exposure to nitroprusside and was mimicked by exposure to 50-500 muM 8-bromo cyclic GMP. 8-Bromoguanosine 5'-monophosphate did not alter (32)P incorporation. Dibutyryl cyclic AMP at 50 muM had no effect upon (32)P incorporation whereas a higher concentration (0.5 mM) caused increased or decreased (32)P incorporation into some, but not all, of the same proteins. 8-Bromo cyclic AMP (5 mM) produced only small changes in (32)P incorporation. The pattern of (32)P incorporation induced by a relatively high concentration of isoproterenol 0.1 mM was similar but not identical to that seen with 0.5 mM dibutyryl cyclic AMP. The present study indicates that the incorporation of (32)P into endogenous proteins of intact rat aorta can be regulated by nitroprusside. These effects can be mimicked by cyclic GMP analogues and only partially by cyclic AMP analogues or isoproterenol. Presumably, these effects of nitroprusside are mediated through a cyclic GMP-dependent process (protein kinase or phosphatase) which may play a role in the relaxant properties of nitroprusside and cyclic GMP.