The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells

Purpose: Thrombin inhibits intercellular CA(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on Myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective Myosin II ATPase inhibitor, was used to investigate whether the effect of Thrombin is mediated by enhanced actomyosin contractility.

Methods: BCECs were exposed to Thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. CA(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated CA(2+) responses.

Results: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the CA(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the Thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the Thrombin effect on paracrine and gap junctional IC. In the absence of Thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [CA(2+)](i) transients in response to Thrombin or ATP.

Conclusions: Blebbistatin prevents the inhibitory effects of Thrombin on intercellular CA(2+) wave propagation. The findings demonstrate that Myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.