Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jun 1;364(Pt 2):431–440. doi: 10.1042/BJ20020191

Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase.

Mitsuo Mita 1, Hayato Yanagihara 1, Shigeru Hishinuma 1, Masaki Saito 1, Michael P Walsh 1
PMCID: PMC1222588  PMID: 12023886

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.

Full Text

The Full Text of this article is available as a PDF (222.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen B. G., Walsh M. P. The biochemical basis of the regulation of smooth-muscle contraction. Trends Biochem Sci. 1994 Sep;19(9):362–368. doi: 10.1016/0968-0004(94)90112-0. [DOI] [PubMed] [Google Scholar]
  2. Benham C. D., Tsien R. W. A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature. 1987 Jul 16;328(6127):275–278. doi: 10.1038/328275a0. [DOI] [PubMed] [Google Scholar]
  3. Bolton T. B., Prestwich S. A., Zholos A. V., Gordienko D. V. Excitation-contraction coupling in gastrointestinal and other smooth muscles. Annu Rev Physiol. 1999;61:85–115. doi: 10.1146/annurev.physiol.61.1.85. [DOI] [PubMed] [Google Scholar]
  4. Burnstock G. The fifth Heymans memorial lecture-Ghent, February 17, 1990. Co-transmission. Arch Int Pharmacodyn Ther. 1990 Mar-Apr;304:7–33. [PubMed] [Google Scholar]
  5. Colburn J. C., Michnoff C. H., Hsu L. C., Slaughter C. A., Kamm K. E., Stull J. T. Sites phosphorylated in myosin light chain in contracting smooth muscle. J Biol Chem. 1988 Dec 15;263(35):19166–19173. [PubMed] [Google Scholar]
  6. Donoso M. V., Steiner M., Huidobro-Toro J. P. BIBP 3226, suramin and prazosin identify neuropeptide Y, adenosine 5'-triphosphate and noradrenaline as sympathetic cotransmitters in the rat arterial mesenteric bed. J Pharmacol Exp Ther. 1997 Aug;282(2):691–698. [PubMed] [Google Scholar]
  7. Eto M., Ohmori T., Suzuki M., Furuya K., Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem. 1995 Dec;118(6):1104–1107. doi: 10.1093/oxfordjournals.jbchem.a124993. [DOI] [PubMed] [Google Scholar]
  8. Feng J., Ito M., Kureishi Y., Ichikawa K., Amano M., Isaka N., Okawa K., Iwamatsu A., Kaibuchi K., Hartshorne D. J. Rho-associated kinase of chicken gizzard smooth muscle. J Biol Chem. 1999 Feb 5;274(6):3744–3752. doi: 10.1074/jbc.274.6.3744. [DOI] [PubMed] [Google Scholar]
  9. Fukata Y., Amano M., Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci. 2001 Jan;22(1):32–39. doi: 10.1016/s0165-6147(00)01596-0. [DOI] [PubMed] [Google Scholar]
  10. Gailly P., Gong M. C., Somlyo A. V., Somlyo A. P. Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle. J Physiol. 1997 Apr 1;500(Pt 1):95–109. doi: 10.1113/jphysiol.1997.sp022002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gohla A., Schultz G., Offermanns S. Role for G(12)/G(13) in agonist-induced vascular smooth muscle cell contraction. Circ Res. 2000 Aug 4;87(3):221–227. doi: 10.1161/01.res.87.3.221. [DOI] [PubMed] [Google Scholar]
  12. Gong M. C., Cohen P., Kitazawa T., Ikebe M., Masuo M., Somlyo A. P., Somlyo A. V. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. J Biol Chem. 1992 Jul 25;267(21):14662–14668. [PubMed] [Google Scholar]
  13. Gong M. C., Fuglsang A., Alessi D., Kobayashi S., Cohen P., Somlyo A. V., Somlyo A. P. Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem. 1992 Oct 25;267(30):21492–21498. [PubMed] [Google Scholar]
  14. Gong M. C., Iizuka K., Nixon G., Browne J. P., Hall A., Eccleston J. F., Sugai M., Kobayashi S., Somlyo A. V., Somlyo A. P. Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1340–1345. doi: 10.1073/pnas.93.3.1340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gong M. C., Kinter M. T., Somlyo A. V., Somlyo A. P. Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. J Physiol. 1995 Jul 1;486(Pt 1):113–122. doi: 10.1113/jphysiol.1995.sp020795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gunst S. J., al-Hassani M. H., Adam L. P. Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle. Am J Physiol. 1994 Mar;266(3 Pt 1):C684–C691. doi: 10.1152/ajpcell.1994.266.3.C684. [DOI] [PubMed] [Google Scholar]
  17. Hartshorne D. J., Ito M., Erdödi F. Myosin light chain phosphatase: subunit composition, interactions and regulation. J Muscle Res Cell Motil. 1998 May;19(4):325–341. doi: 10.1023/a:1005385302064. [DOI] [PubMed] [Google Scholar]
  18. Himpens B., Matthijs G., Somlyo A. V., Butler T. M., Somlyo A. P. Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle. J Gen Physiol. 1988 Dec;92(6):713–729. doi: 10.1085/jgp.92.6.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirata K., Kikuchi A., Sasaki T., Kuroda S., Kaibuchi K., Matsuura Y., Seki H., Saida K., Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem. 1992 May 5;267(13):8719–8722. [PubMed] [Google Scholar]
  20. Ichikawa K., Ito M., Hartshorne D. J. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem. 1996 Mar 1;271(9):4733–4740. doi: 10.1074/jbc.271.9.4733. [DOI] [PubMed] [Google Scholar]
  21. Ishikawa T., Chijiwa T., Hagiwara M., Mamiya S., Saitoh M., Hidaka H. ML-9 inhibits the vascular contraction via the inhibition of myosin light chain phosphorylation. Mol Pharmacol. 1988 Jun;33(6):598–603. [PubMed] [Google Scholar]
  22. Ito S., Kume H., Honjo H., Katoh H., Kodama I., Yamaki K., Hayashi H. Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol. 2001 Jun;280(6):L1218–L1224. doi: 10.1152/ajplung.2001.280.6.L1218. [DOI] [PubMed] [Google Scholar]
  23. Itoh T., Kubota Y., Kuriyama H. Effects of a phorbol ester on acetylcholine-induced Ca2+ mobilization and contraction in the porcine coronary artery. J Physiol. 1988 Mar;397:401–419. doi: 10.1113/jphysiol.1988.sp017008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jiang M. J., Morgan K. G. Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle. Pflugers Arch. 1989 Apr;413(6):637–643. doi: 10.1007/BF00581814. [DOI] [PubMed] [Google Scholar]
  25. Karaki H., Urakawa N., Kutsky P. Potassium-induced contraction in smooth muscle. Nihon Heikatsukin Gakkai Zasshi. 1984 Dec;20(6):427–444. doi: 10.1540/jsmr1965.20.427. [DOI] [PubMed] [Google Scholar]
  26. Kimura K., Ito M., Amano M., Chihara K., Fukata Y., Nakafuku M., Yamamori B., Feng J., Nakano T., Okawa K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase) Science. 1996 Jul 12;273(5272):245–248. doi: 10.1126/science.273.5272.245. [DOI] [PubMed] [Google Scholar]
  27. Kitazawa T., Gaylinn B. D., Denney G. H., Somlyo A. P. G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem. 1991 Jan 25;266(3):1708–1715. [PubMed] [Google Scholar]
  28. Kitazawa T., Masuo M., Somlyo A. P. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9307–9310. doi: 10.1073/pnas.88.20.9307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kitazawa T., Somlyo A. P. Desensitization and muscarinic re-sensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle. Biochem Biophys Res Commun. 1990 Nov 15;172(3):1291–1297. doi: 10.1016/0006-291x(90)91589-k. [DOI] [PubMed] [Google Scholar]
  30. Kitazawa T., Takizawa N., Ikebe M., Eto M. Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. J Physiol. 1999 Oct 1;520(Pt 1):139–152. doi: 10.1111/j.1469-7793.1999.00139.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kubota Y., Nomura M., Kamm K. E., Mumby M. C., Stull J. T. GTP gamma S-dependent regulation of smooth muscle contractile elements. Am J Physiol. 1992 Feb;262(2 Pt 1):C405–C410. doi: 10.1152/ajpcell.1992.262.2.C405. [DOI] [PubMed] [Google Scholar]
  32. Kuriyama H., Kitamura K., Itoh T., Inoue R. Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels. Physiol Rev. 1998 Jul;78(3):811–920. doi: 10.1152/physrev.1998.78.3.811. [DOI] [PubMed] [Google Scholar]
  33. Lundberg J. M. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol Rev. 1996 Mar;48(1):113–178. [PubMed] [Google Scholar]
  34. Masuo M., Reardon S., Ikebe M., Kitazawa T. A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase. J Gen Physiol. 1994 Aug;104(2):265–286. doi: 10.1085/jgp.104.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Missiaen L., De Smedt H., Droogmans G., Himpens B., Casteels R. Calcium ion homeostasis in smooth muscle. Pharmacol Ther. 1992 Nov;56(2):191–231. doi: 10.1016/0163-7258(92)90017-t. [DOI] [PubMed] [Google Scholar]
  36. Mita M., Walsh M. P. alpha1-Adrenoceptor-mediated phosphorylation of myosin in rat-tail arterial smooth muscle. Biochem J. 1997 Nov 1;327(Pt 3):669–674. doi: 10.1042/bj3270669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nagumo H., Sasaki Y., Ono Y., Okamoto H., Seto M., Takuwa Y. Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am J Physiol Cell Physiol. 2000 Jan;278(1):C57–C65. doi: 10.1152/ajpcell.2000.278.1.C57. [DOI] [PubMed] [Google Scholar]
  38. Nishimura J., Kolber M., van Breemen C. Norepinephrine and GTP-gamma-S increase myofilament Ca2+ sensitivity in alpha-toxin permeabilized arterial smooth muscle. Biochem Biophys Res Commun. 1988 Dec 15;157(2):677–683. doi: 10.1016/s0006-291x(88)80303-6. [DOI] [PubMed] [Google Scholar]
  39. Noda M., Yasuda-Fukazawa C., Moriishi K., Kato T., Okuda T., Kurokawa K., Takuwa Y. Involvement of rho in GTP gamma S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Lett. 1995 Jul 3;367(3):246–250. doi: 10.1016/0014-5793(95)00573-r. [DOI] [PubMed] [Google Scholar]
  40. Ono T., Mita M., Suga O., Hashimoto T., Oishi K., Uchida M. K. Receptor-coupled shortening of alpha-toxin-permeabilized single smooth muscle cells from the guinea-pig stomach. Br J Pharmacol. 1992 Jul;106(3):539–543. doi: 10.1111/j.1476-5381.1992.tb14371.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Racchi H., Irarrázabal M. J., Howard M., Morán S., Zalaquett R., Huidobro-Toro J. P. Adenosine 5'-triphosphate and neuropeptide Y are co-transmitters in conjunction with noradrenaline in the human saphenous vein. Br J Pharmacol. 1999 Mar;126(5):1175–1185. doi: 10.1038/sj.bjp.0702396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rembold C. M., Murphy R. A. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res. 1988 Sep;63(3):593–603. doi: 10.1161/01.res.63.3.593. [DOI] [PubMed] [Google Scholar]
  43. Saitoh M., Ishikawa T., Matsushima S., Naka M., Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem. 1987 Jun 5;262(16):7796–7801. [PubMed] [Google Scholar]
  44. Sato K., Leposavic R., Publicover N. G., Sanders K. M., Gerthoffer W. T. Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester. J Physiol. 1994 Dec 15;481(Pt 3):677–688. doi: 10.1113/jphysiol.1994.sp020473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Senba S., Eto M., Yazawa M. Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle. J Biochem. 1999 Feb;125(2):354–362. doi: 10.1093/oxfordjournals.jbchem.a022294. [DOI] [PubMed] [Google Scholar]
  46. Somlyo A. P., Himpens B. Cell calcium and its regulation in smooth muscle. FASEB J. 1989 Sep;3(11):2266–2276. doi: 10.1096/fasebj.3.11.2506092. [DOI] [PubMed] [Google Scholar]
  47. Somlyo A. P., Somlyo A. V. Signal transduction and regulation in smooth muscle. Nature. 1994 Nov 17;372(6503):231–236. doi: 10.1038/372231a0. [DOI] [PubMed] [Google Scholar]
  48. Somlyo A. P., Somlyo A. V. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol. 2000 Jan 15;522(Pt 2):177–185. doi: 10.1111/j.1469-7793.2000.t01-2-00177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Somlyo A. V., Somlyo A. P. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharmacol Exp Ther. 1968 Jan;159(1):129–145. [PubMed] [Google Scholar]
  50. Suematsu E., Resnick M., Morgan K. G. Change of Ca2+ requirement for myosin phosphorylation by prostaglandin F2 alpha. Am J Physiol. 1991 Aug;261(2 Pt 1):C253–C258. doi: 10.1152/ajpcell.1991.261.2.C253. [DOI] [PubMed] [Google Scholar]
  51. Swärd K., Dreja K., Susnjar M., Hellstrand P., Hartshorne D. J., Walsh M. P. Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol. 2000 Jan 1;522(Pt 1):33–49. doi: 10.1111/j.1469-7793.2000.0033m.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Takata Y., Kato H. Effects of Ca antagonists on the norepinephrine release and contractile responses of isolated canine saphenous veins to high KCl. Jpn J Pharmacol. 1985 Apr;37(4):381–394. doi: 10.1254/jjp.37.381. [DOI] [PubMed] [Google Scholar]
  53. Tansey M. G., Hori M., Karaki H., Kamm K. E., Stull J. T. Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle. FEBS Lett. 1990 Sep 17;270(1-2):219–221. doi: 10.1016/0014-5793(90)81272-p. [DOI] [PubMed] [Google Scholar]
  54. Uehata M., Ishizaki T., Satoh H., Ono T., Kawahara T., Morishita T., Tamakawa H., Yamagami K., Inui J., Maekawa M. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997 Oct 30;389(6654):990–994. doi: 10.1038/40187. [DOI] [PubMed] [Google Scholar]
  55. Weber L. P., Seto M., Sasaki Y., Swärd K., Walsh M. P. The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle. Biochem J. 2000 Dec 1;352(Pt 2):573–582. [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES