Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta
Inhibition of protein kinase C-mediated contraction by Rho kinase inhibitor fasudil in rabbit aorta
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DOI：
10.1007/s00210-004-0975-9
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发表时间：
2004-10
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影响因子：
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通讯作者：
Erika Shimomura;M. Shiraishi;T. Iwanaga;M. Seto;Y. Sasaki;M. Ikeda;Katsuaki Ito
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文献类型：
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作者：
Erika Shimomura;M. Shiraishi;T. Iwanaga;M. Seto;Y. Sasaki;M. Ikeda;Katsuaki Ito
蛋白激酶C（PKC）通过佛波酯激活，通过抑制MLC磷酸酶（MLCP）增加肌球蛋白轻链（MLC 20）磷酸化，并增强血管平滑肌的收缩。我们研究了Rho激酶，这是已知的抑制MLCP，是否参与MLC 20磷酸化引起的佛波酯，12-脱氧佛波13-异丁酸酯（DPB），在兔牙周炎。DPB（1 μM）增加MLC 20磷酸化和张力。Rho激酶抑制剂法舒地尔（10 μM）抑制DPB诱导的收缩，并降低MLC 20在Ser 19（MLC激酶磷酸化位点）的磷酸化，但不影响总MLC 20的磷酸化。用不含Ca 2+的EGTA溶液冲洗65.4 mM KCl收缩的主动脉，可引起MLC 20快速去磷酸化和舒张。当DPB存在于冲洗液中时，MLC 20的去磷酸化和松弛被抑制。在本实验中，PKC抑制剂Ro 31 -8220（10 μM）可抑制DPB诱导的总MLC 20和Ser 19的磷酸化。法舒地尔也抑制Ser 19磷酸化至与Ro 31 -8220相似的程度，并加速松弛，其小于Ro 31 -8220引起的松弛。磷脂酶A2抑制剂ONO-RS-082（5 μM）抑制DPB诱导的Ser 19磷酸化，但仅短暂降低张力，提示花生四烯酸参与了磷酸化，存在MLC 20磷酸化非依赖性机制。当法舒地尔与ONO-RS-082组合时，法舒地尔对张力产生额外的抑制，而不进一步抑制Ser 19磷酸化。DPB使MLCP的130 kDa肌球蛋白结合亚基（MBS）磷酸化，法舒地尔抑制这种磷酸化。这些数据表明，法舒地尔抑制DPB诱导的收缩和MLC 20在MLC激酶靶向位点的磷酸化是Rho激酶抑制的结果。因此，PKC依赖的血管平滑肌的Ca 2+敏化涉及Rho激酶。一个MLC 20磷酸化独立的机制也参与了Ca ~（2+）敏化。
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 MLC20phosphorylation caused by a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), in rabbit aortas. DPB (1 μM) increased MLC20phosphorylation and tension. The Rho kinase inhibitor fasudil (10 μM) inhibited the DPB-induced contraction and decreased the MLC20phosphorylation 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 MLC20and relaxation. When DPB was present in the rinsing solution, the MLC20dephosphorylation and the relaxation were inhibited. In this protocol, Ro31-8220 (10 μM), a PKC inhibitor, suppressed the phosphorylation of total MLC20and 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 A2inhibitor 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 MLC20phosphorylation-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 MLC20at 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 MLC20phosphorylation-independent mechanism is also involved in the Ca2+-sensitization.