Cell Signaling Induced by Activation of Purinergic Receptors in Bovine Corneal Endothelial Cells

Abstract

Abstract: : Purpose: The activation of the purinergic receptors is associated with enhanced ion transport, volume regulation, and barrier function in the corneal endothelium. This study has examined cell signaling induced by the activation of the purinergic receptors in cultured bovine corneal endothelial cells (BCEC). Methods: The focus is on acute activation PKA, PKC, and MAPK (ERK1/2) mediated pathways in response to P1/P2 agonists and their subsequent convergence on myosin light chain (MLC) phosphorylation. The phosphorylated and unphosphorylated MLC were separated by urea-glycerol gel and identified by Western blotting. Expressions of P2 receptor subtypes and an ecto-apyrase (CD39) were determined by RT-PCR. Activity of P1 purinergic receptors was determined indirectly by the phosphorylation of cAMP responsive element binding protein (pCREB) by Western blotting. The agonists were exposed in the presence of 500 µM IBMX. ERK1/2 phosphorylation was assessed by Western blotting. ATP release was measured by the luciferase-luciferrin bioluminescence technique. Results: (1) Exposure to adenosine (ADO; 200 µM; 30 min) and NECA (50 µM; 30 min), A2B-sensitive P1 agonists, induced phosphorylation of CREB similar to forskolin (30 µM; 60 min). These agents also led to dephosphorylation of MLC at similar concentrations/duration of exposure. ADO and NECA also induced ERK1/2 phosphorylation which was prevented by U0126 (10 µM; 30 min), a MAPK inhibitor. ADO induced MLC dephosphorylation was partially inhibited by pre-exposure to U0126. (2) Positive bands for the expression of mRNA for P2Y1 and P2Y2 subtypes as well as CD39 (an ecto-apyrase) was found in BCEC. ATP (100 µM, 10 min), a non-selective agonist for P2Y receptors, induced ERK1/2 phoshorylation which was prevented by pre-exposure to U0126. ATP (15 min) induced MLC dephosphorylation. Acute hypotonic shock, which led to ATP release, stimulated ERK1/2 phosphorylation. Exposure to PMA (100 nM) for short (15 min) and long (24 hrs) periods led to phosphorylation and desphosphorylation of MLC, respectively. Conclusions. (1) MLC phosphorylation was inhibited by PKA and activated by MAPK, (2) PKA induced MLC dephosphorylation underlies the reduced paracellular permeability in response to ADO and its analogs (Riley et al. IOVS, 39(11) 1998), and (3) ATP induced net dephosphorylation of MLC could be through ADO formation by ectoapyrases or direct activation of protein phosphatases (Noll et al., AJP Cell, 279, C717, 2000)

Keywords: adenosine • cornea: endothelium • ion transporters