ATP–dependent Paracrine Intercellular Communication in Corneal Endothelial Cells

Abstract

Abstract: : Purpose:Gap junctional intercellular communication (GJIC) and paracrine IC (PIC) are two forms of IC, which is critical for coordinated responses to extracellular stresses. Usually, the paracrine signaling molecule in PIC is ATP. This study has investigated IC in corneal endothelial (CE) cells in response to localized compressive stresses, which also occur during phacoemulsification due to cavitational forces caused by bubble implosions. Methods: Cultured bovine CE (BCEC), grown on glass coverslips, were used for all experiments. IC was examined through Ca²⁺ wave propagation which was initiated by indentation of a single cell (referred to as mechanical stimulus or MS) with a glass micropipette (∼1 µm tip) driven by a Piezo actuator. The wave propagation was recorded by a confocal laser scanning fluorescence microscope using the [Ca²⁺]_i–sensitive dye Fluo–4. The expression of the ATP–sensitive P2 purinergic receptors and ecto–ATPases was examined by RT–PCR. Results:Upon indentation, the [Ca²⁺]_i–sensitive fluorescence increased to a peak (F_P) in < 600 ms. This peak was followed by a slow decay towards the baseline (i.e., intensity before MS) in < 170 s. Subsequently, the neighboring (NB) cells showed a similar pattern of fluorescence rise but with distance–dependent time lags. The wave extended up to five NB cells with the percentage of responsive cells (defined as cells with F_P greater than 110% of their respective baselines) reducing significantly with increasing distance from the MS cell. Similarly, F_P in NB cells also decreased with increasing distance. Cells that were not connected to MS and NB cells also showed a fluorescence rise. Application of ARL–67156 (an ecto–ATPase inhibitor, 100 µM) enhanced the wave propagation, while an inhibitory effect was seen in the presence of non–selective P2Y antagonist suramin (100 µM), exposure to apyrase VI and VII (10 U/mL), or U–73122 (PLC–ß inhibitor; 10 µM). The intercellular wave propagation was insensitive to SKF–96365 (inhibitor of capacitative calcium entry; 50 µM). RT–PCR showed expression of mRNA for P2Y1, P2Y2, and CD39 (an ecto–ATPase) in BCEC. Conclusions: CE exhibits Ca²⁺ wave propagation in response to mechanical indentation. Results of experiments with ARL, suramin, apyrase and experiments with disconnected cells, taken together, suggest that the wave propagation is mediated to a significant extent by ATP–dependent PIC.

Keywords: calcium • cell–cell communication • ion channels