Research Paper
Subject Category: Gastrointestinal pharmacology
British Journal of Pharmacology (2008) 153, 1214–1224; doi:10.1038/sj.bjp.0707686; published online 21 January 2008
Contractile agonists attenuate cGMP levels by stimulating phosphorylation of cGMP-specific PDE5; an effect mediated by RhoA/PKC-dependent inhibition of protein phosphatase 1
K S Murthy1
1Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA
Correspondence: Dr KS Murthy, Department of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, 1101 E marshall Street, Richmond, Virginia 23298, USA. E-mail: skarnam@vcu.edu
Received 27 September 2007; Revised 7 December 2007; Accepted 12 December 2007; Published online 21 January 2008.
Abstract
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 
M) 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.
Keywords:
phosphodiesterases, protein kinase G, protein phosphatase, muscle relaxation
Abbreviations:
GSNO, S-nitrosoglutathione; 8-pCPT-cGMPS, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate; KT5823, (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizobenzo9a,g)cycloocta(c,d,e)-trinden-1-one; PP1, protein phosphatase 1
