摘要
Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca2 + ([Ca2 +]i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca2 +]i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 渭M, reduced the PM fluidity by activating PKC伪 and PKC尾II translocation to PM and also the ER fluidity directly. In accordance, 3 渭M AA did not impact the basal [Ca2 +]i but significantly suppressed the thapsigargin-induced Ca2 + release and Ca2 + influx. Inhibition of PKC with G枚6983 or knockdown of PKC伪 or PKC尾 using shRNA significantly attenuated the inhibitory effects of 3 渭M AA on PM fluidity and agonist-induced Ca2 + signal. However, AA at high concentration, 30 渭M, caused robust release and entry of Ca2 + accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKC尾I and PKC尾II redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 渭M AA-induced Ca2 + influx by 45%, Gd3 + at concentration of 10 渭M could completely abolish both release and entry of Ca2 + induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca2 + signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca2 +]i response, while Gd3 +-sensitive 鈥榩ores鈥?in PM/ER play an important role in AA-induced Ca2 + signal in HEK293 cells.