Thrombin-Induced Inter-Endothelial Gap Formation in Bovine Corneal Endothelial Cells

Abstract

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 G_q/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