Contractile agonists attenuate cGMP levels by stimulating phosphorylation of cGMP-specific PDE5; an effect mediated by RhoA/PKC-dependent inhibition of protein phosphatase 1

Background and purpose: 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. Experimental approach: 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. Key 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 μM) 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. Conclusion and implications: 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. British Journal of Pharmacology (2008) 153, 1214–1224; doi:10.1038/sj.bjp.0707686; published online 21 January 2008