ANF elicits phosphorylation of the cGMP phosphodiesterase is vascular smooth muscle cells

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur, Vascular smooth muscle cells (VSMC) were used to determine if PDES is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubatcd with P-32(i) to label cellular ATP and then treated with atrial natriuretic factor (ANF). in the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation ofthe 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic Monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDES, had no effect on PDES phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two-to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PT)ES, demonstrated no ANF-depenctent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunonoprecipitated PDE5 from these cells will purified PKG, cGMP, and a phosphorylation mixture containing [gamma-P-32]ATP resulted in P-32(i) incorporation into PDES that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.