卡巴胆碱通过大鼠膀胱平滑肌细胞中肌球蛋白磷酸酶靶向亚基1（MYPT1）的苏氨酸696磷酸化诱导信号传导。
Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells.
作者信息
Liu Benchun, Lee Yung-Chin, Alwaal Amjad, Wang Guifang, Banie Lia, Lin Ching-Shwun, Lin Guiting, Lue Tom F
机构信息
Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, 400 Parnassus Ave., Ste A-610, San Francisco, CA, 94143-0738, USA.
Department of Urology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
出版信息
Int Urol Nephrol. 2016 Aug;48(8):1237-1242. doi: 10.1007/s11255-016-1303-2. Epub 2016 Apr 27.
PURPOSE
Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs).
MATERIALS AND METHODS
Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot.
RESULTS
In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100 μM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test).
CONCLUSIONS
Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.
目的
有证据表明，Rho相关蛋白激酶（ROCK）介导的肌球蛋白磷酸酶靶向亚基1（MYPT1）磷酸化在平滑肌收缩中起核心作用。然而，ROCK催化的膀胱平滑肌中MYPT1第696位苏氨酸磷酸化的生理意义仍存在争议。我们试图直接观察大鼠膀胱平滑肌细胞（RBMSCs）中卡巴胆碱给药后Rho A/ROCK的定量蛋白表达以及MYPT1第696位苏氨酸的磷酸化情况。
材料与方法
从大鼠膀胱中获取原代培养的平滑肌细胞。通过蛋白质印迹法评估Rho A/ROCK的表达以及MYPT1第696位苏氨酸的磷酸化情况，研究毒蕈碱激动剂卡巴胆碱诱导的浓度和时间进程效应。
结果
在剂量进程研究中，基于蛋白质印迹结果，卡巴胆碱在15 - 100 μM浓度范围内使MYPT1第696位苏氨酸的磷酸化（p-MYPT1）显著增加（p < 0.05，方差分析检验）。在时间进程研究中，用15 μM卡巴胆碱处理细胞，从3至15小时显著增强了p-MYPT1的表达（p < 0.05，方差分析检验），并在10至120分钟诱导了Rho A的表达（p < 0.05，方差分析检验）。
结论
卡巴胆碱可诱导ROCK通路的表达，导致MYPT1第696位苏氨酸磷酸化，从而使RBMSCs持续收缩。
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