Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2 + signaling in HEK293 cells

Abstract

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, 3μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3μM AA did not impact the basal [Ca2+]i but significantly suppressed the thapsigargin-induced Ca2+ release and Ca2+ influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3μM AA on PM fluidity and agonist-induced Ca2+ signal. However, AA at high concentration, 30μM, caused robust release and entry of Ca2+ accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30μM AA-induced Ca2+ influx by 45%, Gd3+ at concentration of 10μM 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 ‘pores’ in PM/ER play an important role in AA-induced Ca2+ signal in HEK293 cells.