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This Article Full Text Full Text (PDF) All Versions of this Article: 297/6/F1534    most recent 00095.2009v1 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 PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Wang, T. Articles by Moreland, R. S. PubMed PubMed Citation Articles by Wang, T. Articles by Moreland, R. S.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Departments of ¹Pharmacology and Physiology and ; ²Pathology and Laboratory Medicine, Drexel University College of Medicine, Philadelphia, Pennsylvania

Submitted February 18, 2009 ; accepted in final form September 28, 2009

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr³⁸-CPI-17 and Thr⁶⁹⁶/Thr⁸⁵⁰-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr³⁸-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr⁸⁵⁰ phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr⁸⁵⁰-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

MLC phosphatase; myosin phosphatase-targeting subunit; CPI-17; bisindolylmaleimide-1; H-1152; MLC phosphorylation