| Research Abstract |
We demonstrated that intracellular CaィイD12+ィエD1 concentration ([CaィイD12+ィエD1]ィイD2iィエD2) was increased in a restricted region with a diameter of less than a few μm in lens epithelial cells loaded with fluo-4 when the cells were stimulated mechanically in the presence of lysophosphatidic acid (LPA), an intercellular phospholipid messenger. The CaィイD12+ィエD1 response was inhibited by lowering extracelluar CaィイD12+ィエD1 concentration, but not by thapsigargin, suggesting that the CaィイD12+ィエD1 response is a CaィイD12+ィエD1 influx event. The CaィイD12+ィエD1 increase reached to the peak level in the starting region within 100 ms and concentrically diffused in a restricted region. We named the CaィイD12+ィエD1 influx "CaィイD12+ィエD1 spots". In order to clarify the spatiotemporal characteristics of CaィイD12+ィエD1 spots, we visualized CaィイD12+ィエD1 spot phenomenon using a high-speed three-dimensional confocal system with a piezoelectric driven objective, which permits simultaneous imaging of focal and apical plan
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es of lens epithelial cells at each 37.6 ms interval. CaィイD12+ィエD1 spots in the apical plane was more clearly visualized than that in the focal plane, suggesting that CaィイD12+ィエD1 spots reflect CaィイD12+ィエD1 influx on the apical membrane. From these results, we propose a hypothesis that CaィイD12+ィエD1 spots is an elementary CaィイD12+ィエD1 influx event underlying localized cellular responses to mechanical stress. Furthermore, the involvement of Rho-mediated signal transduction and integrin signaling in CaィイD12+ィエD1 spots were investigated. Pretreatment of Clostridium botulinum exoenzyme C3 transferase, an inhibitor of Rho protein, or cytochalasin D, which depolymerizes the actin cytoskeleton, blocked formation of stress fibers, but not affect the CaィイD12+ィエD1 spots. Additionally, genistein, an inhibitor of tyrosine kinase including pp125ィイD1FAKィエD1, did not inhibit the CaィイD12+ィエD1 spots. On the other hand, GRGDSP, an inhibitor of integirn, partially inhibited the CaィイD12+ィエD1 spots. These results suggest that LPA enhances the mechanical stress-induced CaィイD12+ィエD1 spots by modulating recognizing site of integirn to RGD sequence, but not by formation of actin stress fibers. Less
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