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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ increase. 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, secondary translocation was delayed, occurring after the Ca²⁺ wave; however, this 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 from extracellular space through MS channel is required. Our results support the implication of PKCα in the Ca²⁺ signaling pathway in response to mechanical stress in ECs.