The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.
- cyclic nucleotides
- protein kinase A
- protein kinase G
- soluble guanylate cyclase
Abbreviations used: PDE, phosphodiesterase; GC, guanylate cyclase; AC, adenylate cyclase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; SNP, sodium nitroprusside; PKI, protein kinase inhibitor; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated kinase kinase; PKC, protein kinase C; VIP, vasoactive intestinal peptide; PACAP, pituitary AC-activating peptide; IBMX, isobutylmethylxanthine; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; cBIMPS, Sp-5,6-dichloro-1-β-d-ribofuranosyl benzimidazole 3′,5′-cyclic monophosphothioate; NOS, NO synthase; ECL® (Amersham Pharmacia Biotech), enhanced chemiluminescence.
- The Biochemical Society, London ©2001