Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells

Purpose: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the 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 immunoblotting. Elevation of cAMP in response to agonists of A(2b) receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity. Results: Exposure to ADO (200 mum; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 mum; 30 min), known agonists of A(2b) receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 pm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 mum ADO. CGS-21680, a selective A(2a) agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 muM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm(2). Conclusions: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity. (C) 2004 Elsevier Ltd. All rights reserved.