Visualization of elementary mechanosensitive Ca2+-influx events, Ca2+ spots, in bovine lens epithelial cells

Local increases in the intracellular Ca2+ concentration ([Ca2+]i) in several regions within the bovine lens epithelial cell during application of mechanical stress were clearly visualized in the presence of lysophosphatidic acid (LPA), a bioactive lysophospholipid, using real-time confocal microscopy. We called the phenomenon 'Ca2+ spots'. Ca2+ spots started in a circular area with a radius of about 1.5 m. These Ca2+ spots spread concentrically, resulting in a mean global increase in [Ca2+]i. The local increase often occurred in a stepwise manner or repetitively at the same region. The spatiotemporal properties of the Ca2+ spots were completely different from those of the Ca2+ wave induced by ATP, a Ca2+-mobilizing agonist. Ca2+ spots were inhibited by decreasing the extracellular Ca2+ concentration or by the presence of Gd3+, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, suggesting that Ca2+ spots arise from Ca2+ influx through Gd3+-sensitive MS channels. On the assumption that, in lens epithelial cells, the open probability of the MS channel is 0.4, the membrane potential is 56 mV and the channel conductance is 50 pS, the estimated maximum flux of Ca2+ in a Ca2+ spot (0.4 x 10-17 to 4.7 x 10-17 mol x s(-1)) was comparable to currents of one or a few MS channels. On real-time three-dimensional confocal imaging analysis, which permitted simultaneous imaging of basal and apical planes of cells at 37.6 ms intervals, Ca2+ spots on the apical plane were more clearly visualized than those on the basal plane. From these results, we propose that the Ca2+ spot is an elementary Ca2+-influx event through MS channels directly coupled with the first step in mechanoreception In addition, our results strongly suggest that LPA functions as an endogenous factor affecting mechanotransduction systems.