Adenosine Induces Dephosphorylation of Myosin Light Chain in Corneal Endothelial Cells

Abstract

Abstract: : Purpose. Dephosphorylation of Myosin II regulatory light chain (MLC) promotes cellular barrier integrity through relaxation of the cortical actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC). Methods. MLC phosphorylation was assessed by urea–glycerol gel electrophoresis and western blotting. Phosphorylation of the cAMP responsive element binding protein (CREB), determined by western blotting, was used to confirm the elevation of cAMP in response to ADO and other agonists for A2B receptors. Activation of ERK1/2 was assessed by western blotting. Results. Exposure to ADO (200 µM; 30 min) and NECA (50 µM; 30 min), known agonists of A2B receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 µM; 30 min). Exposure to ADO, NECA, and FSK also led to dephosphorylation of MLC by 51%, 42%, and 47%, respectively. ADO–induced dephosphorylation was both dose– and time–dependent, with maximum dephosphorylation noticeable at 18 min. As much as 31% dephosphorylation was observed with 1 µM ADO. Selective A2A agonist CGS–21680 had little effect on MLC dephosphorylation. ADO also induced ERK1/2 activation (phosphorylation) which could be prevented by U0126 (10 µM; 30 min; MAP kinase kinase–specific inhibitor). NECA and FSK also induced ERK1/2 activation similar to ADO. U0126 inhibited MLC phosphorylation significantly under basal conditions (17%). ADO–induced MLC dephosphorylation was enhanced by concomitant exposure to U0126 (25% more dephosphorylation). Conclusions. (1) CREB phosphorylation in response to ADO and NECA, indicating activation of the cAMP–PKA axis, suggests that BCEC express the A2B receptors. (2) ERK1/2 is also activated by cAMP in BCEC and it promotes MLC phosphorylation. However, activation of the cAMP–PKA axis in BCEC overwhelms ERK1/2–mediated response leading to net MLC dephosphorylation presumably through competing pathways involving inactivation of MLCK and/or ROCK–1.

Keywords: pharmacology • pump/barrier function • ion channels