Inhibitory phosphorylation of soluble guanylyl cyclase by muscarinic m2 receptors via Gbetagamma-dependent activation of c-Src kinase.
In gastrointestinal smooth muscle, cGMP levels in response to relaxant agonists are regulated by activation of phosphodiesterase 5 and inhibition of soluble guanylyl cyclase (sGC) in a feedback mechanism via cGMP-dependent protein kinase. The aim of the present study was to determine whether contractile agonists modulate cGMP levels by cross-regulating sGC activity. In gastric muscle cells, acetylcholine (ACh) stimulated Src activity and induced sGC phosphorylation. Concurrent stimulation of cells with ACh attenuated sGC activity and cGMP formation in response to the nitric oxide (NO) donor, S-nitrosoglutathione (GSNO). The effect of ACh on Src activity, sGC phosphorylation, and on GSNO-stimulated sGC activity and cGMP formation were blocked by the m2 receptor antagonist (methoctramine), pertussis toxin, and by inhibitors of phosphatidylinositol 3 kinase, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], or Src kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, in dispersed muscle cells and in cells expressing Galpha(i) minigene or Gbetagamma-scavenging peptide, whereas the m3 receptor antagonist, N-(2-chloroethyl)-4-piperidinyl diphenylacetate, or expression of the Galpha(q) minigene had no effect. ACh also attenuated sGC activity and cGMP formation in response to the NO-independent activator, YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzylindazole]. The pattern implied that phosphorylation of sGC by c-Src kinase inhibits NO-sensitive sGC activity, and the inhibition was not due to a decrease in the binding of NO but probably due to decrease in catalytic activity. We conclude that cGMP levels are cross-regulated by contractile agonists via a mechanism that involves c-Src-dependent phosphorylation of sGC, leading to inhibition of sGC activity and cGMP formation. The finding highlights a novel mechanism for attenuation of the NO/sGC/cGMP signal by G(i)-coupled contractile agonists, in addition to their inhibitory effect on adenylyl cyclase and cAMP formation.
Murthy
Karnam S
KS
Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. skarnam@hsc.vcu.edu
D000818:Animals; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle; D010766:Phosphorylation; D011817:Rabbits; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D013270:Stomach; D019061:src-Family Kinases / Q000378:metabolism*
D043585:Receptor, Muscarinic M2; D018160:Receptors, Cytoplasmic and Nuclear; D019061:src-Family Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase