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Rho-Kinase-Mediated Diphosphorylation of Myosin Regulatory Light Chain is a Unique Biochemical Mechanism in Human Uterine Myocytes Open Access


Other title
In-cell Western
Myosin Regulatory Light Chain
Type of item
Degree grantor
University of Alberta
Author or creator
Aguilar, Hector N
Supervisor and department
Mitchell, Bryan F. (Obstetrics and Gynecology)
Examining committee member and department
Kassiri, Zamaneh (Physiology)
Lopez Bernal, Andres (University of Bristol)
Davidge, Sandra (Obstetrics and Gynecology)
Brindley, David (Biochemistry)
Department of Physiology

Date accepted
Graduation date
Doctor of Philosophy
Degree level
Rationale: Smooth muscle (SM) contraction results from activation of the cellular contractile machinery by phosphorylation of myosin regulatory light chain (RLC) at S19 (pRLC) by RLC kinase (MLCK). Subsequent phosphorylation at T18 yields diphosphorylated-RLC (ppRLC), which has been shown to enhance the myosin ATPase activity, and therefore might have implications for the ability of the muscle to generate tension. In uterine myocytes, the extent of diphosphorylation is greater than that observed in vascular myocytes. In preliminary experiments using cultured human uterine smooth muscle cells (hUSMC), we observed that rho-kinase (ROK) inhibition caused a marked reduction in ppRLC, but surprisingly, not pRLC. Therefore we pursued a quantitative comparison in ROK-mediated phosphorylation of RLC in hUSMC and human vascular myocytes (hVSMC). Objectives: 1) to develop and validate quantitative methods for assessment of phosphorylated RLC, and 2) to apply these methods in studies comparing ROK-mediated phosphorylation of RLC in hUSMC and hVSMC. Findings: We have developed and validated two assays for quantitative measurements of phosphorylated RLC using phospho-specific antibodies: 1) a high-throughput in-cell western assay, and 2) western blotting-based quantification of RLC-phospho-states after separation with phos-tag SDS-PAGE. These techniques revealed that hUSMC differ from hVSMC in their primary phosphorylation responses to stimulation. hUSMC respond to oxytocin with increased pRLC and ppRLC, whereas hVSMC respond to endothelin-1 with increased pRLC only. In hUSMC, only ppRLC was sensitive to ROK inhibition using a pharmacologic inhibitor, and this occurred at approximately 100-fold lower concentration of ROK inhibitor than that required to achieve an equivalent reduction of pRLC in hVSMC. Conclusions: In hUSMC, ROK phosphorylates RLC at T18 downstream of MLCK-mediated phosphorylation at S19. This sequential phosphorylation of RLC forms ppRLC. This role of ROK activity appears unique to hUSMC and is a fundamental difference from hVSMC. These findings suggest the possibility of a uterine-specific therapeutic avenue for prevention or arrest of preterm labor achieved by targeting ROK to reduce ppRLC levels in hUSMC. Our data also suggest that ROK inhibition might avoid the prohibitive side effects of currently used agents for preterm labour, of which the most common and serious is hypotension that results from unwanted vascular relaxation.
License granted by Hector Aguilar ( on 2011-08-16T02:40:52Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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