Abstract
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.
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Acknowledgments
Research reported in this publication was also supported by NIDDK of the National Institutes of Health under award number R01 DK069655 and R56 DK105097. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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All the authors have declared no conflict of interest.
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This study was approved by the Institutional Animal Care and Use Committee of University of California San Francisco.
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Benchun Liu and Yung-Chin Lee have contributed equally to this work.
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Liu, B., Lee, YC., Alwaal, A. et al. Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells. Int Urol Nephrol 48, 1237–1242 (2016). https://doi.org/10.1007/s11255-016-1303-2
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DOI: https://doi.org/10.1007/s11255-016-1303-2