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1From the Laboratory of Physiology, KULeuven, Campus Gasthuisberg, Leuven, Belgium; and the 2Indiana University, School of Optometry, Bloomington, Indiana.
PURPOSE. Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs).
METHODS. A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (
1 µm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence.
RESULTS. BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 ± 3,200 µm2 (N = 21). The percentage of responsive cells (defined as cells showing maximum NF > 1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 µM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 µM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 µM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs.
CONCLUSIONS. PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell–cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.
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