Contractile agonists attenuate cGMP levels by stimulating phosphorylation of cGMP-specific PDE5; an effect mediated by RhoA/PKC-dependent inhibition of protein phosphatase 1.
OBJECTIVE
In gastrointestinal smooth muscle cGMP levels in response to relaxant agonists are regulated by PKG-mediated phosphorylation and activation of phosphodiesterase 5 (PDE5). The aim of the present study was to determine whether contractile agonists modulate cGMP levels by cross-regulating PDE5 activity and to identify the mechanism of action.


METHODS
Dispersed and cultured muscle cells from rabbit stomach were treated with the nitric oxide donor, S-nitrosoglutathione (GSNO), or with a contractile agonist, ACh and GSNO. PDE5 phosphorylation and activity, and cGMP levels were determined.


RESULTS
GSNO stimulated PDE5 phosphorylation and activity and increased cGMP levels in gastric smooth muscle cells. Concurrent activation of cells with ACh augmented GSNO-stimulated PDE5 phosphorylation and activity, and attenuated cGMP levels. The effect of ACh was blocked by the m3 receptor antagonist and by inhibitors of protein kinase C (PKC) or RhoA, but not by the m2 receptor antagonist or inhibitors of PI hydrolysis. The effects of ACh on PDE5 phosphorylation and activity, and cGMP levels were mimicked by a low concentration of tautomycin (10 nM), and a high (1 microM) but not low (1 nM) concentration of okadaic acid. PDE5 was associated with protein phosphatase 1 (PP1) and dephosphorylated by the catalytic subunit of PP1 but not PP2A.


CONCLUSIONS
In gastrointestinal smooth muscle cGMP levels are cross-regulated by contractile agonists via a mechanism that involves RhoA-dependent, PKC-mediated inhibition of PP1 activity. This leads to augmentation of PDE5 phosphorylation and activity, and inhibition of cGMP levels.
Murthy
K S
KS
Department of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA. skarnam@vcu.edu
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D043587:Receptor, Muscarinic M3 / Q000819:agonists* / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000494:pharmacology*; D013270:Stomach / Q000166:cytology; D020742:rhoA GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism
D043587:Receptor, Muscarinic M3; D026422:S-Nitrosoglutathione; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D020742:rhoA GTP-Binding Protein; D006152:Cyclic GMP; D000109:Acetylcholine