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Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta

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Abstract

Protein kinase C (PKC) activation by a phorbol ester increases myosin light chain (MLC20) phosphorylation through inhibition of MLC phosphatase (MLCP) and enhances contraction of vascular smooth muscle. We investigated whether Rho kinase, which is known to inhibit MLCP, is involved in the MLC20 phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 μM) increased MLC20 phosphorylation and tension. The Rho kinase inhibitor fasudil (10 μM) inhibited the DPB-induced contraction and decreased the MLC20 phosphorylation at Ser19, a site phosphorylated by MLC kinase, although it did not affect the phosphorylation of total MLC20. Rinsing a 65.4 mM KCl-contracted aorta with Ca2+-free, EGTA solution caused rapid dephosphorylation of MLC20 and relaxation. When DPB was present in the rinsing solution, the MLC20 dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 μM), a PKC inhibitor, suppressed the phosphorylation of total MLC20 and Ser19 induced by DPB. Fasudil also inhibited the Ser19 phosphorylation to a degree similar to Ro31-8220 and accelerated relaxation, which was less than the relaxation caused by Ro31-8220. The phospholipase A2 inhibitor ONO-RS-082 (5 μM) inhibited the DPB-induced Ser19 phosphorylation but only transiently decreased the tension, suggesting the involvement of arachidonic acid in the phosphorylation and the existence of a MLC20 phosphorylation-independent mechanism. When fasudil was combined with ONO-RS-082, fasudil exerted additional inhibition of the tension without further inhibition of the Ser19 phosphorylation. DPB phosphorylated the 130 kDa myosin binding subunit (MBS) of MLCP and fasudil inhibited the phosphorylation. These data suggest that the inhibition by fasudil of DPB-induced contraction and phosphorylation of MLC20 at the MLC kinase-targeted site is a result of inhibition of Rho kinase. Thus, the PKC-dependent Ca2+-sensitization of vascular smooth muscle involves Rho kinase. A MLC20 phosphorylation-independent mechanism is also involved in the Ca2+-sensitization.

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Authors and Affiliations

  1. Department of Veterinary Pharmacology, Faculty of Agriculture, Miyazaki University, Miyazaki, 889-2192, Japan

    Erika Shimomura, Mitsuya Shiraishi, Takahiro Iwanaga, Masahiro Ikeda & Katsuaki Ito

  2. Second Laboratory for Pharmacology, Institute for Life Science Research, Asahi Kasei Pharma Corporation, Fuji, Shizuoka, 416-8501, Japan

    Minoru Seto

  3. Department of Pharmacology, Kitasato University Pharmacy School, Tokyo, 108-8641, Japan

    Yasuharu Sasaki

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  1. Erika Shimomura
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  2. Mitsuya Shiraishi
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  3. Takahiro Iwanaga
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  4. Minoru Seto
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  5. Yasuharu Sasaki
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  6. Masahiro Ikeda
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  7. Katsuaki Ito
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Correspondence to Katsuaki Ito.

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Shimomura, E., Shiraishi, M., Iwanaga, T. et al. Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta. Naunyn-Schmiedeberg's Arch Pharmacol 370, 414–422 (2004). https://doi.org/10.1007/s00210-004-0975-9

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  • DOI: https://doi.org/10.1007/s00210-004-0975-9

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