Gap Junctional Intercellular Communication in Bovine Corneal Endothelial Cells (BCEC)

Abstract

Purpose: : Intercellular communication (IC) in non–excitable cells is mediated through gap junctions (GJIC) and/or through release of paracrine mediators (paracrine IC or PIC) such as adenosine triphosphate (ATP). This study was conducted to investigate the contribution of gap junctions (GJs) towards IC as represented by intercellular propagation of Ca²⁺ waves in cultured bovine corneal endothelial cells (BCECs)

Methods: : An intercellular Ca²⁺ wave was elicited by applying a point mechanical stimulus (PMS) to a single cell in a monolayer. Changes in [Ca²⁺]_i were visualized using the fluorescent dye Fluo–4. The area reached by the Ca²⁺ wave, called the active area (AA), was determined as a measure of efficacy of IC. Connexin–43 (Cx43) expression was investigated by RT–PCR and immunocytochemistry. GJIC was assessed by fluorescence recovery after photobleaching (FRAP), Lucifer yellow (LY) dye transfer after scrape–loading, and pharmacological inhibition of GJIC. ATP release in response to PMS was measured using luciferin–luciferase bioluminescence protocol.

Results: : BCECs showed expression of Cx43, a major Cx subtype, at mRNA and protein levels. GJIC was evident from dye transfer between cells in scrape–loading and FRAP experiments. Gap27 (300 µM), a connexin mimetic peptide that blocks GJs formed by Cx43, reduced the fluorescence recovery in FRAP experiments by 19% (N = 37). Gap27 also reduced AA of the PMS–induced Ca²⁺ wave from 73,689 µm² (N = 36) to 26,936 µm² (N = 39), implying that GJIC contribution to the spread of the wave is at least ∼ 63%. PIC inhibitors, 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 AA by 91% (N = 21), 67% (N = 11) and 55% (N = 24), respectively. These data suggest that GJIC contributes no more than ∼ 9% towards AA of the Ca²⁺ wave. Gap27 did not affect ARL–67156 (200 µM; inhibitor of ectonucleotidases)–induced enhancement in AA, PMS–induced ATP release, and zero [Ca²⁺]_o–induced LY 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 Ca²⁺ wave. The residual AA after inhibition of PIC by apyrases was much smaller than the reduction of AA by Gap27, indicating interaction between GJIC and PIC.

Conclusions: : Functional GJs are present in BCECs. Both GJIC and PIC contribute significantly to IC, and the two pathways interact.

Keywords: cornea: endothelium • gap junctions/coupling • cell-cell communication