Abstract
Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 +/− and Rock2 +/− mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.
Similar content being viewed by others
Abbreviations
- ACh:
-
Acetylcholine
- eNOS:
-
Endothelial nitric oxide synthase
- ET-1:
-
Endothelin-1
- GAPDH:
-
Glyceraldehyde 3-phosphate dehydrogenase
- MLC:
-
Myosin light chain
- MLCK:
-
MLC kinase
- MLCP:
-
MLC phosphatase
- MYPT1:
-
Myosin phosphatase targeting subunit 1
- PDBu:
-
Phorbol 12,13-dibutyrate
- PE:
-
Phenylephrine
- ROCK:
-
Rho-associated kinase (Rho-kinase)
- SNP:
-
Sodium nitroprusside
References
Arner A, Lofgren M, Morano I (2003) Smooth, slow and smart muscle motors. J Muscle Res Cell Motil 24:165–173
Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P (2007) The selectivity of protein kinase inhibitors: a further update. Biochem J 408:297–315
Brown JH, Del Re DP, Sussman MA (2006) The Rac and Rho hall of fame: a decade of hypertrophic signaling hits. Circ Res 98:730–742
De Mey JGR, Uitendaal MP, Boonen HCM, Vrijdag MJJF, Daemen MJAP, Struyker-Boudier HAJ (1989) Acute and long-term effects of tissue culture on contractile reactivity in renal arteries of the rat. Circ Res 65:1125–1135
Dimopoulos G, Semba S, Kitazawa K, Eto M, Kitazawa T (2007) Ca2+-dependant rapid Ca2+ sensitization of contraction in arterial smooth muscle. Circ Res 100:121–129
Félétou M, Vanhoutte PM (2006) Endothelial dysfunction: a multifaceted disorder. Am J Physiol 291:H985–H1002
Gerthoffer WT (2007) Mechanisms of vascular smooth muscle cell migration. Circ Res 100:607–621
Kitazawa T, Eto M, Woodsome TP, Khalequzzaman M (2003) Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. J Physiol 546:879–889
Kitazawa T, Semba S, Huh YH, Kitazawa K, Eto M (2009) Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1. J Physiol 587:3587–3603
Lehoux S, Esposito B, Merval R, Tedgui A (2005) Differential regulation of vascular focal adhesion kinase by steady stretch and pulsatility. Circulation 111:643–649
Lindqvist A, Nordström I, Malmqvist U, Nordenfelt P, Hellstrand P (1999) Long-term effects of Ca2+ on structure and contractility of vascular smooth muscle. Am J Physiol 277:C64–C73
Löhn M, Plettenburg O, Ivashchenko Y, Kannt A, Hofmeister A, Kadereit D, Schaefer M, Linz W, Kohlmann M, Herbert JM, Janiac P, O’Connor SE, Ruetten H (2009) Pharmacological characterization of SAR407899, a novel rho-kinase inhibitor. Hypertension 54:676–683
Loirand G, Guérin P, Pacaud P (2006) Rho kinases in cardiovascular physiology and pathophysiology. Circ Res 98:322–334
Merrilees MJ, Scott L (1982) Organ culture of rat carotid artery: maintenance of morphological characteristics and of pattern of matrix synthesis. In Vitro 18:900–910
Mukai H (2003) The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC. J Biochem 133:17–27
Murata T, Suzuki N, Yamawaki H, Sato K, Hori M, Karaki H, Ozaki H (2005) Dexamethasone prevents impairment of endothelium-dependent relaxation in arteries cultured with fetal bovine serum. Eur J Pharmacol 515:134–141
Noma K, Oyama N, Liao JK (2006) Physiological role of ROCKs in the cardiovascular system. Am J Physiol 290:C661–C668
Noma K, Rikitake Y, Oyama N, Yan G, Alcaide P, Liu P-Y, Wang H, Ahl D, Sawada N, Okamoto R, Okamoto R, Hiroi Y, Shimizu K, Luscinskas FW, Sun J, Liao JK (2008) ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury. J Clin Invest 118:1632–1644
Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:767–801
Ozaki H, Karaki H (2002) Organ culture as a useful method for studying the biology of blood vessels and other smooth muscle tissues. Jpn J Pharmacol 89:93–100
Sasaki Y, Suzuki M, Hidaka H (2002) The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol Ther 93:225–232
Schwartz SM (1997) Smooth muscle migration in atherosclerosis and restenosis. J Clin Invest 99:2814–2816
Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H, Oshima M, Noda Y, Matsumura F, Taketo MM, Narumiya S (2005) ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J Cell Biol 168:941–953
Shimokawa H, Rashid M (2007) Development of Rho-kinase inhibitors for cardiovascular medicine. Trends Pharmacol Sci 28:296–302
Shimokawa H, Takeshita A (2005) Rho-kinase is an important therapeutic target in cardiovascular medicine. Arterioscler Thromb Vasc Biol 25:1767–1775
Thorne GD, Paul RJ (2003) Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor. Am J Physiol 284:C999–C1005
Thumkeo D, Keel J, Ishizaki T, Hirose M, Nonomura K, Oshima H, Oshima M, Taketo MM, Narumiya S (2003) Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death. Mol Cell Biol 23:5043–5055
Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389:990–994
Velasco G, Armstrong C, Morrice N, Frame S, Cohen P (2002) Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett 527:101–104
Woodsome TP, Polzin A, Kitazawa K, Eto M, Kitazawa T (2006) Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells. J Cell Sci 119:1769–1780
Acknowledgments
We thank Drs. James Sherley and Albert Wang of the BBRI for their comments on the manuscript. This work was supported by National Institute of Health grant R01 HL070881 to TK and HL052233, HL080187, and DK085006 to JKL.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Huh, Y.H., Zhou, Q., Liao, J.K. et al. ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery. J Muscle Res Cell Motil 32, 65–76 (2011). https://doi.org/10.1007/s10974-011-9252-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10974-011-9252-y