AbstractWe observed the kinetics of protein kinase Cα (PKCα) and the intracellular Ca2+wave in endothelial cells (ECs) in response to microscopic mechanical stress to investigate the effect of mechanical stress on PKCα translocation. The results show that a focal mechanical stimulus induced biphasic and directional PKCα translocation; PKCα initially translocated toward distinct spots near or at the membrane and then accumulated at the stimulus point. The low initial translocation occurred simultaneously in parallel with the increase in Ca2+. Initial translocation was inhibited in spite of Ca2+increase when the diacylglycerol (DAG) binding domain of PKCα was inhibited, suggesting that translocation requires intracellular Ca2+increase and DAG. On the other hand, high secondary translocation was delayed, occurring after the Ca2+wave; however, this secondary translocation occurred even when Ca2+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 Ca2+influx through MS channels is required. Our results support the implication of PKCα in the Ca2+signaling pathway in response to mechanical stress in ECs.Summary statementIn response to a focal mechanical stimulus, PKCα in an endothelial cell was initially translocated toward distinct spots near or at the membrane and then accumulated at the stimulus point.