Role of regulatory mechanism of myosin phosphatase in stretch-induced contraction of vascular smooth muscle

对牵张诱导的犬基底动脉肌球蛋白轻链(MLC)磷酸化进行了研究。在四乙基铵(5 Mm)存在下，在15min的刺激时间内，以1 mm/s的速度缓慢拉伸至初始肌肉长度的1.5倍，可增加多个磷酸化的MLC物种(单磷酸化、二磷酸化和三磷酸化物种)。肌球蛋白轻链激酶抑制剂ML-9和蛋白激酶C抑制剂Calphostin C可部分抑制MLC的这种多重磷酸化，但可被ML-9和Rho-Kinase抑制剂Calphostin C-Y-27632所阻断，几乎完全抑制MLC的磷酸化。Stretch增加肌球蛋白磷酸酶130 kDa调节亚基(类型1，MBS)的磷酸化，导致肌球蛋白磷酸酶的抑制。Y-27632抑制MBS的磷酸化。在4种PKC亚型(PKC_α，δ，ζ和η)中，只有PKC_α在15min拉伸时从胞浆转移到膜部分。经典蛋白激酶C的抑制剂Go6976可抑制MLC的多重磷酸化，而蛋白激酶C的特异性抑制剂5μM rotlerin不能抑制δ的多重磷酸化。3-μ-M冈田酸(OA)在此浓度下抑制磷酸酶2A的活性，可减弱80 mM KCl2诱发的犬基底动脉收缩。这种衰减可以被Go6976抵消，但不能被roterin抵消。OA产生了多个磷酸化的MLC物种，并且磷酸化模式与STRAND产生的相似。OA对Stretch产生的磷酸化模式没有影响。肌动球蛋白ATPase活性受15分钟牵张和OA的抑制。这些结果提示，Rho/Rho-K途径介导的1型和2A型磷酸酶抑制所揭示的MLCK和PKC的α活性可能参与了牵张诱导的犬基底动脉MLC的多重磷酸化。

Stretch-induced myosin light chain (MLC) phosphorylation in canine basilar artery was investigated. In the presence of tetraethylammonium (5 mM), slow stretch at a rate of 1 mm/sec up to 1.5 times initial muscle length during a stimulus period of 15 min produced an increase in multiple phosphorylated MLC species (mono-, di- and tri-phosphorylated species). Although this multiple phosphorylation of MLC was partly inhibited by ML-9, an inhibitor of myosin light chain kinase, and calphostin C, a protein kinase C (PKC) inhibitor, it was abolished by ML-9 plus calphostin C. Y-27632, a Rho-kinase inhibitor, almost completely inhibited MLC phosphorylation. Stretch increased the phosphorylation of 130 kDa regulatory subunit of myosin phosphatase (type 1, MBS), resulting the inhibition of myosin phosphatase. Y-27632 inhibited the phosphorylation of MBS. Of 4 PKC isoforms (PKC_α, δ,ζand η), only PKC_α was translocated from the cytosol to the membrane fraction by 15-min stretch. Multiple phosphorylation of MLC was inhibited by Go6976, an inhibitor of classical PKC, but not 5 μM rottlerin, a specific inhibitor of PKCδ. On the other hand, 3μM okadaic acid (OA), which inhibited phosphatase 2A activity at this concentration, attenuated 80 mM KCl-induced contraction of canine basilar artery. This attenuation was countered by Go6976, but not by rotterin. OA produced the multiple phosphorylated MLC species and phosphorylation pattern was similar to that produced by stretch. OA had no effect on the phosphorylation pattern produced by stretch. Actomyosin ATPase activity was inhibited by 15-min stretch and OA. These results suggest than MLCK and PKCα activities unmasked by the inhibition of phosphatases type 1 and type 2A which mediated by Rho/Rho-kinase pathway may be involved in stretch-induced multiple phosphorylation of MLC in the canine basilar artery.