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

• Author / Creator

  Aguilar, Hector N

• 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.

• Subjects / Keywords

  • Uterus
  • In-cell Western
  • Myosin Regulatory Light Chain
  • Myometrium
  • Phos-tag
  • Rho-Kinase
  • RhoA
  • Phosphorylation
  • MYPT1
  • Endothelin-1
  • Contractility
  • Oxytocin

• Graduation date

  Fall 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3113N

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.