Modulation of soluble guanylate cyclase activity by phosphorylation.
The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.
Murthy
Karnam S
KS
Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University Richmond, Richmond, VA 23298-0711, USA. skarnam@hsc.vcu.edu
D002227:Carbazoles / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011863:Radioimmunoassay; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012995:Solubility; D000071756:Soluble Guanylyl Cyclase; D013270:Stomach / Q000166:cytology / Q000201:enzymology; D013873:Thionucleotides / Q000494:pharmacology
D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D020030:Nitric Oxide Donors; D018160:Receptors, Cytoplasmic and Nuclear; D013873:Thionucleotides; C073601:KT 5823; C083763:8-bromoguanosino-3',5'-cyclic monophosphorothioate; D009599:Nitroprusside; D019319:Okadaic Acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP