ANF ELICITS PHOSPHORYLATION OF THE CGMP PHOSPHODIESTERASE IS VASCULARSMOOTH-MUSCLE CELLS

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific pho sphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) i n vitro. Binding of cGMP to the allosteric sites of PDE5 is required f or 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 phosphoryla ted 93-kDa protein band was immunoprecipitated from early passaged pri mary cultures of VSMC that had been preincubatcd with P-32(i) to label cellular ATP and then treated with atrial natriuretic factor (ANF). i n the absence of ANF, there was no detectable incorporation of radiola beled phosphate into this band. Phosphorylation ofthe 93-kDa protein w as augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic M onophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcG MP, which interacts poorly with the allosteric sites of PDES, had no e ffect on PDES phosphorylation in the absence of ANF. Phosphorylation o f PDE5 in response to treatment of cells with ANF was associated with a two-to fourfold increase in PDE activity in immunoprecipitates. Mult iple-passaged VSMC, which are deficient in PKG but retain PT)ES, demon strated no ANF-depenctent increase in phosphorylation or catalytic act ivity of PDE5. However, incubation of immunonoprecipitated PDE5 from t hese cells will purified PKG, cGMP, and a phosphorylation mixture cont aining [gamma-P-32]ATP resulted in P-32(i) incorporation into PDES tha t 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 regu lation.