Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase.
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 Thr(38)-CPI-17 and Thr(696)/Thr(850)-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr(38)-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 Thr(850) 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 Thr(850)-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.
Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.
Tanchun
T
Wang
Derek M
DM
Kendig
Elaine M
EM
Smolock
Robert S
RS
Moreland
D000818:Animals; D002217:Carbachol / Q000494:pharmacology*; D018679:Cholinergic Agonists / Q000494:pharmacology*; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism; D009129:Muscle Tonus / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1 / Q000378:metabolism; D021122:Protein Subunits; D011817:Rabbits; D001743:Urinary Bladder / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C491521:CPI-17 protein, Oryctolagus cuniculus; D018679:Cholinergic Agonists; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D002217:Carbachol; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D054645:Protein Phosphatase 1