OBSERVATION OF MEMBRANE LIPID IN BOVINE AORTIC ENDOTHELIAL CELL UNDER MECHANICAL STIMULUS
Satoshi Miyamoto, Masataka Arai, Kazuhiro Nakashima, Toshihiro Sera, Susumu Kudo
Kyushu University, Japan
Intracellular signaling occurs from G-protein coupled receptor (GPCR) in plasma membrane. Once GPCR receives extracellular stimulus, phospholipase C (PLC) is activated and hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and diacylglycerol (DAG). These processes are carried out at the plasma membrane, particularly PIP2 and DAG are known as membrane lipids. IP3 diffused into cytoplasm and activates Ca2+ store, which leads to increase the intracellular concentrations of Ca2+. DAG and Ca2+ are the activators of protein kinase C (PKC) family. On the other hand, the cellular sensing system to mechanical stimulus have been reported. For example, increase the intracellular concentrations of Ca2+ and PLC activation by poking stimulus. However, the response system has not been understood.
In this study, we focused on the PIP2 hydrolysis on the membrane to investigate the intracellular response as cellular sensing system to mechanical stimulus. To achieve this objective, we observed the activations of PIP2 and DAG in bovine aortic endothelial cells (BAECs) when poking stimulus was exposed. For observation of the membrane lipids in living cells, the fluorescent plasmids were transfected in BAECs. GFP-C1-PKCgamma-C1A and GFP-C1-PLCdelta-PH were used for DAG and PIP 2 imaging, respectively. In experiment, the micropipette needle with tip radius of 1µm was used for poking stimulus to living cell, and the fluorescent change of the microscopic images was analyzed.
The fluorescent distribution change of GFP-C1PKCgamma-C1A was observed in overall stimulated cell, which indicates DAG expression at the whole plasma membrane. Previous study reports that PKCα translocation to stimulated point. DAG is necessary for PKCα translocation, however in this study the localized DAG distribution was not observed. These results suggested that DAG expression may have no relationship to PKCα translocation.