May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Thrombin-Induced Inter-Endothelial Gap Formation in Bovine Corneal Endothelial Cells
Author Affiliations & Notes
  • M. Satpathy
    Sch of Optometry, Indiana Univ, Bloomington, IN, United States
  • P. Gallagher
    School of Medicine, Physiology, Indiana Univ, Indianapolis, IN, United States
  • S.P. Srinivas
    School of Medicine, Physiology, Indiana Univ, Indianapolis, IN, United States
  • Footnotes
    Commercial Relationships  M. Satpathy, None; P. Gallagher, None; S.P. Srinivas, None.
  • Footnotes
    Support  Supported by NIH 11107 (SPS
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3447. doi:
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      M. Satpathy, P. Gallagher, S.P. Srinivas; Thrombin-Induced Inter-Endothelial Gap Formation in Bovine Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3447.

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

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Abstract: : Purpose. In certain epithelia and vascular endothelium, the phosphorylation of Myosin Light Chain (MLC) induces an increase in paracellular permeability through contraction of the cortical actin cytoskeleton. This study presents a robust model for examining the role of MLC phosphorylation and its regulation in corneal endothelium. Methods. Experiments were carried out with primary cultures of bovine corneal endothelium. Expressions of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) isoforms (smooth mucles and non-muscle forms), a key component in the direct-pathway leading to phosphorylation of MLC, were determined by Western blotting. MLC phosphorylation was induced by exposure to thrombin (1 u/mL) and NaF (10 mM) which are known to elicit MLC phoshorylation in a variety of cell types. Phosphorylated and unphosphorylated MLC were separated by urea-glycerol gel and identified by Western blotting. MLCK was inhibited by ML-7 (50 µM; 30 min exposure). Rho kinase, a potent inhibitor of myosin phosphatase, was inhibited by Y27632 (100 µM; 30 min exposure). Altered cytoskeleton was assessed through phalloidin staining of actin microfilaments. Intracellular free Ca2+ was measured by Fura-2-AM in order to identify the specificity of thrombin receptor coupling to G proteins. Results. Thrombin showed Ca2+ rise with and without extracellular Ca2+ indicating coupling to Gq/11 G protein. RT-PCR showed expression of ROCK1, ROCKII, MLCK and RhoA. Two MLCK isoforms, 220 and 130 kd proteins, corresponding to smooth muscle and non-muscle isoforms, respectively were found to be expressed. NaF and thrombin induced MLC phosphorylation independently. Chelerythrine and Y27632, inhibitors of PKC and Rho Kinase, respectively, prevented thrombin-induced MLC phosphorylation significantly. Distinct cortical microfilament organization in cultured cells, found in resting cells, was severely disrupted by thrombin. In fact, thrombin exposure led to extensive inter-endothelial gap formation. Conclusions. Thrombin-induced MLC phosphorylation is dependant on PKC and Rho kinase. Ca2+ rise in response to thrombin is from IP3-sensitive stores. MLC phosphorylation can be expected to affect the barrier function of the corneal endothelium, a significant factor in the pump-leak model of corneal transparency.

Keywords: calcium • cornea: endothelium • signal transduction: pharmacology/physiology 

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