ANF elicits phosphorylation of the cGMP phosphodiesterase in 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 PDE5 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 preincubated with 32(Pi) 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 of the 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 PDE5, had no effect on PDE5 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 PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 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.
Wyatt
T A
TA
Department of Molecular Physiology and Biophysics and of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Naftilan
A J
AJ
Francis
S H
SH
Corbin
J D
JD
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000818:Animals; D001011:Aorta / Q000201:enzymology; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007163:Immunosorbent Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
C016276:8-bromocyclic GMP; D009320:Atrial Natriuretic Factor; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP