title: Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase.
abstract: Depolarization of the sarcolemma of smooth muscle cells activates voltage-gated Ca2+ channels, influx of Ca2+ and activation of cross-bridge cycling by phosphorylation of myosin catalysed by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK). Agonist stimulation of smooth muscle contraction often involves other kinases in addition to MLCK. In the present study, we address the hypothesis that membrane depolarization-induced contraction of rat caudal arterial smooth muscle may involve activation of Rho-associated kinase (ROK). Addition of 60 mM K+ to de-endothelialized muscle strips in the presence of prazosin and propranolol induced a contraction that peaked rapidly and then declined to a steady level of force corresponding to approx. 30% of the peak contraction. This contractile response was abolished by the Ca2+-channel blocker nicardipine or the removal of extracellular Ca2+. An MLCK inhibitor (ML-9) inhibited both the phasic and tonic components of K+-induced contraction. On the other hand, the ROK inhibitors Y-27632 and HA-1077 abolished the tonic component of K+-induced contraction, and slightly reduced the phasic component. Phosphorylation levels of the 20-kDa light chain of myosin increased rapidly in response to 60 mM K+ and subsequently declined to a steady-state level significantly greater than the resting level. Y-27632 abolished the sustained and reduced the phasic elevation of the phosphorylation of the 20-kDa light chain of myosin, without affecting the K+-induced elevation of cytosolic free Ca2+ concentration. These results indicate that ROK activation plays an important role in the sustained phase of K+-induced contraction of rat caudal arterial smooth muscle, but has little involvement in the phasic component of K+-induced contraction. Furthermore, these results are consistent with inhibition of myosin light-chain phosphatase by ROK, which would account for the sustained elevation of myosin phosphorylation and tension in response to membrane depolarization.
authors.affiliation: Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. mitsuom@my-pharm.ac.jp
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Mita
authors.forename: Hayato
authors.initials: H
authors.lastname: Yanagihara
authors.forename: Shigeru
authors.initials: S
authors.lastname: Hishinuma
authors.forename: Masaki
authors.initials: M
authors.lastname: Saito
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D001158:Arteries / Q000201:enzymology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008564:Membrane Potentials; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D054460:rho-Associated Kinases
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; C049347:fasudil; D011188:Potassium