Adenosine Opposes Thrombin-Induced Inhibition of Intercellular Calcium Wave in Corneal Endothelial Cells

Abstract

In corneal endothelial cells, intercellular Ca(2+) waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercellular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells. An intercellular Ca(2+) wave was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. The area of Ca(2+) wave propagation was measured by [Ca(2+)](i) imaging using the fluorescent dye Fluo-4. Gap junctional intercellular communication was assessed by fluorescence recovery after photobleaching. Activity of hemichannels was determined by uptake of the hydrophilic dye Lucifer yellow in a Ca(2+)-free medium containing 2 mM EGTA. Adenosine triphosphate (ATP) release in response to mechanical stimulation was measured using the luciferin-luciferase technique. Gap26, a connexin mimetic peptide, was used to block hemichannels. Exposure to thrombin or TRAP-6 (a selective PAR-1 agonist) inhibited the Ca(2+) wave propagation by 70%. Pretreatment with adenosine prevented this inhibitory effect of thrombin. NECA (a potent A2B agonist) and forskolin, agents known to elevate cAMP in bovine corneal endothelial cells, also suppressed the effect of thrombin. The A1 receptor agonist CPA failed to inhibit the effect of thrombin. Similar to the effects on Ca(2+) wave propagation, adenosine prevented the thrombin-induced reduction in the fluorescence recovery during photobleaching experiments. Furthermore, pretreatment with adenosine prevented both thrombin and TRAP-6 from blocking the uptake of Lucifer yellow in a Ca(2+)-free medium. However, adenosine was ineffective in overcoming the Gap26-mediated block of Lucifer yellow uptake. In consistence with Lucifer yellow uptake through hemichannels, the thrombin-induced inhibition of ATP release was overcome by pretreatment with adenosine. Adenosine prevents thrombin-induced inhibition of hemichannel-mediated paracrine intercellular communication and of gap junctional intercellular communication. The mechanism involves an increase in cAMP, which results in inhibition of RhoA and a subsequent decrease in myosin light chain phosphorylation. Since myosin light chain dephosphorylation causes a decrease in contractility of the actin cytoskeleton, the results suggest possible effects of the actin cytoskeleton on gap junctions and connexin hemichannels.