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This Article Full Text Full Text (PDF) Supplemental Figures All Versions of this Article: 297/2/G361    most recent 00112.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Ihara, E. Articles by MacDonald, J. A. PubMed PubMed Citation Articles by Ihara, E. Articles by MacDonald, J. A.

NEUROREGULATION AND MOTILITY

Ca²⁺-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada

Submitted 20 March 2009 ; accepted in final form 12 June 2009

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC₂₀) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca²⁺-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca²⁺-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC₂₀ (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca²⁺-independent LC₂₀ kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK^(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC₂₀ and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

gastrointestinal smooth muscle; calcium sensitization; myosin phosphatase; CPI-17