Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
J Cell Mol Med. 2012 Dec;16(12):2978-89. doi: 10.1111/j.1582-4934.2012.01625.x.
Phosphorylation of myosin regulatory light chain (RLC) triggers contraction in smooth muscle myocytes. Dephosphorylation of phosphorylated RLC (pRLC) is mediated by myosin RLC phosphatase (MLCP), which is negatively regulated by rho-associated kinase (ROK). We have compared basal and stimulated concentrations of pRLC in myocytes from human coronary artery (hVM), which has a tonic contractile pattern to myocytes from human uterus (hUM), which has a phasic contractile pattern. Our studies reveal fundamental differences between hVM and hUM regarding the mechanisms regulating phosphorylation RLC. Whereas hVM responded to stimulation by phosphorylation of RLC at S19, hUM responded by forming diphosphorylated RLC (at T18 and S19; ppRLC), which, compared to pRLC, causes two to threefold greater activation of myosin ATPase that provides energy to power the contraction. Importantly, the conversion of pRLC to ppRLC is mediated by ROK. In hUM, MLCP has high activity for ppRLC and this is inhibited by ROK through phosphorylation of the substrate targeting subunit (MYPT1) at T853. Inhibitors of ROK significantly reduce contractility in both hVM and hUM. We demonstrated that inhibition of ppRLC in phasic myocytes (hUM) is 100-fold more sensitive to ROK inhibitors than is pRLC in tonic myocytes (hVM). We speculate that these differences in phosphorylation of RLC might reflect evolution of different contractile patterns to perform distinct physiological functions. Furthermore, our data suggest that low concentrations of ROK inhibitors might inhibit uterine contractions with minimal effects on vascular tone, thus posing a novel strategy for prevention or treatment of conditions such as preterm birth.
肌球蛋白调节轻链（RLC）的磷酸化触发平滑肌肌细胞收缩。磷酸化 RLC（pRLC）的去磷酸化由肌球蛋白 RLC 磷酸酶（MLCP）介导，而 MLCP 受到 rho 相关激酶（ROK）的负调控。我们比较了具有紧张性收缩模式的人冠状动脉（hVM）肌细胞和具有时相性收缩模式的人子宫（hUM）肌细胞中基础状态和刺激状态下的 pRLC 浓度。我们的研究揭示了 hVM 和 hUM 之间在调节磷酸化 RLC 的机制方面存在根本差异。虽然 hVM 通过 S19 处 RLC 的磷酸化对刺激作出反应，但 hUM 通过形成二磷酸化 RLC（在 T18 和 S19 处；ppRLC）对刺激作出反应，与 pRLC 相比，ppRLC 使肌球蛋白 ATP 酶的激活增加两到三倍，为收缩提供能量。重要的是，pRLC 向 ppRLC 的转化由 ROK 介导。在 hUM 中，MLCP 对 ppRLC 具有高活性，而 ROK 通过磷酸化底物靶向亚基（MYPT1）上的 T853 抑制 MLCP。ROK 抑制剂显著降低了 hVM 和 hUM 中的收缩性。我们证明，与紧张性肌细胞（hVM）中的 pRLC 相比，时相性肌细胞（hUM）中 ppRLC 的抑制对 ROK 抑制剂的敏感性高 100 倍。我们推测，RLC 磷酸化的这些差异可能反映了不同收缩模式的进化，以执行不同的生理功能。此外，我们的数据表明，ROK 抑制剂的低浓度可能抑制子宫收缩，而对血管张力的影响最小，因此为预防或治疗早产等疾病提供了一种新的策略。
