Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation.
The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the beta subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its beta subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the beta subunit.
Ferrero
R
R
Departmento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.
Rodríguez-Pascual
F
F
Miras-Portugal
M T
MT
Torres
M
M
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020098:Natriuretic Peptide, C-Type / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects
D004791:Enzyme Inhibitors; D020098:Natriuretic Peptide, C-Type; D009599:Nitroprusside; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D006162:Guanylate Cyclase; D006152:Cyclic GMP