title: 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 Ca(2+) ([Ca(2+)](i)) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca(2+)](i) and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3 μM AA did not impact the basal [Ca(2+)](i) but significantly suppressed the thapsigargin-induced Ca(2+) release and Ca(2+) influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3 μM AA on PM fluidity and agonist-induced Ca(2+) signal. However, AA at high concentration, 30 μM, caused robust release and entry of Ca(2+) 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 30 μM AA-induced Ca(2+) influx by 45%, Gd(3+) at concentration of 10 μM could completely abolish both release and entry of Ca(2+) induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca(2+) 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 [Ca(2+)](i) response, while Gd(3+)-sensitive 'pores' in PM/ER play an important role in AA-induced Ca(2+) signal in HEK293 cells.
authors.affiliation: Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, Beijing, PR China.
authors.forename: Lihong
authors.initials: L
authors.lastname: Chen
authors.forename: Qingli
authors.initials: Q
authors.lastname: Meng
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Yu
authors.forename: Chen
authors.initials: C
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Chunying
authors.initials: C
authors.lastname: Cui
authors.forename: Dali
authors.initials: D
authors.lastname: Luo
mesh_terms: D016718:Arachidonic Acid / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D016718:Arachidonic Acid; D011493:Protein Kinase C; D002118:Calcium