May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
P2Y Agonists Induce Myosin Light Chain Dephosphorylation in Corneal Endothelial Cells Through Elevated cAMP
Author Affiliations & Notes
  • K.Y. Tan–Allen
    Optometry, Indiana University, Bloomington, IN
  • M. Satpathy
    Optometry, Indiana University, Bloomington, IN
  • J. Bonanno
    Optometry, Indiana University, Bloomington, IN
  • S. Srinivas
    Optometry, Indiana University, Bloomington, IN
  • Footnotes
    Commercial Relationships  K.Y. Tan–Allen, None; M. Satpathy, None; J. Bonanno, None; S. Srinivas, None.
  • Footnotes
    Support  NIH EY14415 (SPS), EY14415 (SPS), EY08834 (JAB)
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 2203. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      K.Y. Tan–Allen, M. Satpathy, J. Bonanno, S. Srinivas; P2Y Agonists Induce Myosin Light Chain Dephosphorylation in Corneal Endothelial Cells Through Elevated cAMP . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2203.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

Abstract: : Purpose: Phosphorylation of MLC induces actomyosin interaction leading to increased contractility of the actin cytoskeleton. Increased contractility of the cortical actin breaks down the barrier integrity by opposing tethering forces at the level of tight junctions between neighboring cells. This study has investigated the influence of ATP and its P2Y analogs on MLC phosphorylation and barrier integrity in bovine corneal endothelial cells (BCEC). Methods: MLC phosphorylation (% of control, pMLC) was assayed by urea–glycerol gel electrophoresis followed by Western blotting. The barrier integrity was assayed as permeability to horseradish peroxidase (HRP). Since PKA down regulates MLC phosphorylation through cross–talks with MLC kinase (MLCK) and MLC phosphatase (MLCP), changes in [cAMP]i were followed by ELISA. To ascertain whether cAMP elevation is secondary to arachidonic acid metabolites, PGE2 release was also measured by ELISA. Results: (a) ATP (100 µM) promoted MLC dephosphorylation (pMLC = 61.8% at 18 min; n = 9). Pre–exposure to ARL–67156, an 5’ ecto–nucleotidase inhibitor, prevented ATP–induced dephosphorylation. Other P2Y agonists, UTP and ATPγS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. (b) Exposure to P2Y agonists for 15–30 min led to a significant increase in [cAMP]i (about 1.5 – 4 folds compared to untreated controls). The supernatants from the same experiments contained higher levels of PGE2 compared to untreated controls. (c). ATP opposed Rho kinase dependent thrombin–induced MLC phosphorylation. This was comparable to the effects of adenosine/forskolin each of which elevated [cAMP]i by >4–fold. (d) Thrombin–induced increase in HRP flux was suppressed by co–treatment with ATP. Conclusions: Stimulation of P2Y receptors leads to elevated cAMP partly through PGE2 release. In the case of ATP, part of the increase in [cAMP]i is due to its degradation by 5’ecto–nucleotidase to adenosine which activates A2B receptors. Since RhoA/Rho kinase is inhibited by cAMP, we suggest that P2Y agonists–induced MLC dephosphorylation is secondary to elevated cAMP. This conclusion is consistent with the suppression of Rho kinase dependent thrombin–induced MLC phosphorylation and loss of barrier integrity.

Keywords: adenosine • phosphorylation • signal transduction: pharmacology/physiology 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.