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Am J Physiol Gastrointest Liver Physiol 292: G887-G898, 2007. First published December 7, 2006; doi:10.1152/ajpgi.00222.2006
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NEUROREGULATION AND MOTILITY

PKC -isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle

Department of Anatomy and Cell Biology and Centre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia

Submitted 19 May 2006 ; accepted in final form 27 November 2006

PKC is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G protein-coupled receptors. Here, we present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC isoforms, PKC-, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea pig ileum. The tonic component of carbachol-evoked contractions was enhanced by an activator of conventional and novel PKCs, phorbol 12,13-dibutyrate (PDBu; 200 nM or 1 µM), and by an activator of novel PKCs, ingenol 3,20-dibenzoate (IDB; 100 or 500 nM). Enhancement was unaffected by concentrations of bisindolylmaleimide I (BIM-I; 22 nM) that block conventional PKCs or by a PKC--specific inhibitor peptide but was attenuated by higher doses of BIM-I (2.2 µM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC- was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC- immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB, or carbachol resulted in a time- and concentration-dependent translocation of PKC- from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC- in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea pig ileum is mediated through a novel PKC, probably PKC-.

protein kinase C; muscarinic receptor; smooth muscle; cytoskeleton

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