PROREIN KINASE C Α TRANSLOCATION BY MECHANICAL STIMULATION IN SINGLE VASCULAR ENDOTHELIAL CELLS
Masataka Arai, Takumi Hasegawa, Kazuhiro Nakashima, Toshihiro Sera, Susumu Kudo
Kyushu University, Japan
Protein kinase C α (PKCα) is a signaling protein that regulates various cellular functions, including migration, differentiation, and apoptosis. Generally, PKCα is distributed uniformly in the cytoplasm. In addition, previous studies showed that PKCα is translocated to a plasma membrane with pharmacological stimulation.
Intracellular Ca2+ wave regulated several signaltransduction pathways. The translocation of PKCα by pharmacological stimulation occurred with an increase of intercellular Ca2+ concentration. A mechanical stimulation by micropipette also causes a transient rise in intracellular Ca2+ concentration and Ca2+ wave. However, the relationship beween PKCα and Ca2+ wave by mechanical stimulation is not understood well yet.
In this study, we developed an experiment system for simultaneous observation PKCα translocation and Ca2+ wave in single cell in response to mechanical stimulation. Futhermore, we investigated the signaling pathway of PKCα translocation and Ca2+ wave by the mechanical stimulation.
PKCα-Dronpa Green was transfected into bovine aorta endothelial cells by the lipofection reagent HilyMax. Ca2+ was measured using the fluorescent Ca2+ indicator fura-2 AM.
Fluorescence images ware captured at a rate of 146 msec per frame. The single cell was stimulated mechanically with a glass microprobe. The tip of the probe was pulled in less than 3μm in diameter and polished. For investigation of he signaling pathway of PKCα translocation, Go6976 was used for inhibitor of PKCα. Thapsigargin and EGTA were used for deplete of intra- and extracellular Ca2+, respectively.
Mechanical stimulation induced a rapid Ca2+ wave and PKCα translocation to stimulated region. Fluorescence of PKCα reached peak after stimulation in around 20 sec. PKCα translocation was inhibited with Go6976. And, when Ca2+ in the extracellular medium was chelated by EGTA, PKCα translocation was not observed. On the other hand, PKCα translocation was observed with thapsigargin. These results suggested that extracellular Ca2+ is necessary to PKCα translocation by mechanical stimulation.