The corneal endothelium is a monolayer at the posterior surface of the cornea facing the anterior chamber. This monolayer plays an important role in corneal transparency, by acting as a semi-permeable barrier and by regulating the hydration state of the stroma. Thrombin, a serine protease, is known to break down the barrier integrity in corneal endothelial cells and to inhibit intercellular communication (IC). A loss of barrier integrity can occur due to contraction of the actin cytoskeleton, which is induced by phosphorylation of the regulatory light chain of myosin II (MLC). The level of MLC phosphorylation is dynamically regulated by MLCK, PKA, PKC, and Rho kinase secondary to activation of a number of G-protein coupled receptors.

In this study we investigated the mechanism of the effect of thrombin on IC in bovine corneal endothelial cells (BCEC), by studying the effect of pharmacological inhibition of protein kinases and phosphatases that play a role in MLC phosphorylation.

An intercellular Ca²⁺ wave was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca²⁺]_i were imaged by fluorescence microscopy using Fluo-4, and the area reached by the Ca²⁺ wave (active area) was calculated. Gap Junctional IC (GJIC) was assessed by fluorescence recovery after photobleaching (FRAP). Activity of hemichannels was assayed by Lucifer Yellow (LY) uptake and also by ATP release using the Luciferin-Luciferase technique.

Both thrombin and the selective PAR-1 agonist TRAP-6 reduced the active area and also the fluorescence recovery in fluorescence recovery after photobleaching (FRAP) experiments. Pretreatment with ML-7 (MLCK inhibitor), Y-27632 (Rho kinase inhibitor) or chelerythrine (PKC inhibitor) prevented the effect of thrombin on the active area and on the reduction of fluorescence recovery. Absence of a significant thrombin-effect in the presence of the combination of inhibitors of MLCK, Rho kinase and PKC indicate that the major pathways for the effect of thrombin on active area are via these three pathways that are known to alter MLC phosphorylation status.

LY uptake that occurs in Ca²⁺-free medium was inhibited by PAR-1 agonists (thrombin or TRAP-6). This inhibition could be overcome by exposure to drugs which reduce MLC phosphorylation. Thus, when cells were pretreated with ML-7, Y-27632 or chelerythrine, LY uptake was similar to control conditions. These results indicate that LY uptake, which can occur through hemichannels, is blocked by PAR-1 activation by a mechanism involving MLC phosphorylation. We also measured extracellular ATP levels by Luciferin-Luciferase technique. Thrombin markedly reduced ATP release upon mechanical stimulation (median reduction of 69%; N = 55). This decrease in release of ATP is reduced to 23% in the presence of Y-27632 and to 20% in the presence of chelerythrine.

In conclusion, thrombin inhibits intercellular Ca²⁺ wave propagation in BCEC. This effect is due to activation of PAR-1 receptors and involves enhanced MLC phosphorylation by MLCK, PKC- and Rho kinase-sensitive pathways.