PROTEIN-KINASE-A ACTIVITY MODULATES NATRIURETIC PEPTIDE-DEPENDENT CGMP ACCUMULATION IN RENAL-CELLS

The purpose of this work was to examine whether the level of cAMP accu mulation and protein kinase A (PKA) activity influence atrial natriure tic factor (ANF)-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in two renal cell types: rabbit cortical vascular smooth m uscle cells (RCSMC) and SV-40-transformed human glomerular visceral ep ithelial cells (HGVEC-SV1). romocinnamylamino)ethyl]-5-isoquinolinesul fonamide (H-89), a PK4 inhibitor, decreased ANF-stimulated cGMP produc tion in RCSMC in a time- and concentration-dependent manner. ANF stimu lated cGMP production was markedly inhibited after prolonged 9- and 18 -h incubations with 25 mu M H-89 (52 and 65%, respectively) but was no t altered after exposure of cells to this agent for 1 h. 1-(5-Isoquino linylsulfonyl)-2-methylpiperazine and -(2-[methylamino]ethyl)-5-isoqui nolinesulfonamide, protein kinase inhibitors not selective for PKA, di d not reproduce the effect of H-89, even at higher concentrations (50 and 100 mu M). Cycloheximide (10 mu M), a protein synthesis inhibitor, limited the inhibitory effect of H-89, although alone it did not modi fy the ANF-stimulated cGMP production. H-89 did not affect cGMP produc tion when it was stimulated by SIN-1, a nitric oxide donor. Prolonged incubation (18 h) with 8-bromo cAMP or cholera toxin, an activator of G(s) protein resulting in adenylate cyclase stimulation, enhanced ANF- dependent cGMP production by 225 and 176%, respectively. This stimulat ory effect was blocked by 25 mu M H-89. I-125-ANF binding to RCSMC at 4 degrees C was not affected by preincubation of the cells with H-89. There was a 44% decrease in the expression of ANF C receptors measured as the ANF-(4-23)-displaceable I-125-ANF binding at 37 degrees C, whi ch could not, however, explain the inhibitory effect of H-89 on cGMP p roduction. Modulation of ANF- and C-type natriuretic peptide-dependent cGMP production by H-89 and cholera toxin was also found in HGVEC-SV1 with the same characteristics as in RCSMC. Taken together, these resu lts suggest that PKA activity controls the function of natriuretic pep tide guanylate cyclase-coupled receptors in the two cell types studied . PKA-dependent inhibition of a negatively regulatory protein distinct from the receptor itself seems necessary for a full cGMP response.