Gap junctional intercellular communication in bovine corneal endothelial cells

Abstract

Gap junctions and/or paracrine mediators, such as ATP, mediate intercellular communication (IC) in non-excitable cells. This study investigates the contribution of gap junctions toward IC during propagation of Ca 2+ waves in cultured bovine corneal endothelial cells (BCEC) elicited by applying a point mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca 2+] i were visualized using the fluorescent dye Fluo-4. The area reached by the Ca 2+ wave, called the active area (AA), was determined as a measure of efficacy of IC. RT–PCR and Western blotting showed expression of Cx43, a major form of connexin, in BCEC. In scrape-loading (using lucifer yellow) and fluorescence recovery after photobleaching (FRAP; using carboxyfluorescein) protocols, significant dye transfer of the hydrophilic dyes was evident indicating functional gap junctional IC (GJIC) in BCEC. Gap27 (300 μM), a connexin mimetic peptide that blocks gap junctions formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19%. Gap27 also reduced the active area of the Ca 2+ wave induced by point mechanical stimulation from 73,689 μm 2 to 26,936 μm 2, implying that GJIC contribution to the spread of the wave is at least ∼63%. Inhibitors of ATP-mediated paracrine IC (PIC), such as a combination of apyrase VI and apyrase VII (5 U/ml each; exogenous ATPases), suramin (200 μM; P2Y antagonist), or Gap26 (300 μM; blocker of Cx43 hemichannels) reduced the active area by 91%, 67%, and 55%, respectively. Therefore, estimating the contribution of GJIC from the residual active area after PIC inhibition appears to suggest that GJIC contributes no more than ∼9% towards the active area of the Ca 2+ wave. Gap27 did not affect the enhancement in active area induced by ARL-67156 (200 μM, ectonucleotidase inhibitor), ATP release induced by point mechanical stimulation, and zero [Ca 2+] o-induced lucifer yellow uptake, indicating that the peptide has no influence on PIC. Exposure to Gap27 in the presence of PIC inhibitors led to a significant further inhibition of the Ca 2+ wave. The finding that the residual active area after inhibition of PIC by apyrases was much smaller than the reduction of the active area by Gap27, provides evidence for interaction between GJIC and PIC. These findings together suggest that functional gap junctions are present in BCEC, that both GJIC and PIC contribute significantly to IC, and that the two pathways interact.