Spatial and temporal translocation of PKCα in single endothelial cell in response to mechanical stimulus.

Endothelial cells (ECs) are exposed to various environmental forces, and a Ca wave is occurred in mechanical stimulated cells. Pharmacological studies reveal that the translocation of protein kinase Cα (PKCα) to the membrane is observed simultaneously with intracellular Ca wave. In this study, we investigate whether and how the kinetics of PKCα in ECs is induced in response to mechanical stress. The results show that a mechanical stimulus induced biphasic and directional PKCα translocation; PKCα initially translocated near or at the membrane and then accumulated at the stimulus point. The initial translocation occurred simultaneously with Ca increase. Initial translocation was inhibited in spite of Ca increase when the diacylglycerol (DAG) binding domain of PKCα was inhibited, suggesting that translocation requires intracellular Ca increase and DAG. On the other hand, secondary translocation was delayed, occurring after the Ca wave; however, this translocation occurred even when Ca release from the endoplasmic reticulum was inhibited, while it did not occur when the mechanosensitive (MS) channel was inhibited. These results indicated that at least Ca influx from extracellular space through MS channel is required. Our results support the implication of PKCα in the Ca signaling pathway in response to mechanical stress in ECs.