EFFECT OF LINOLEIC-ACID HYDROPEROXIDE ON ENDOTHELIAL-CELL CALCIUM HOMEOSTASIS AND PHOSPHOLIPID HYDROLYSIS

The relationship between intracellular free calcium ion concentrations ([Ca2+]i) and release of arachidonic acid from membrane phospholipids following peroxidation was examined in rabbit aortic endothelial cell s treated with linoleic acid hydroperoxide (LOOH). LOOH (0.1-0.4) mu m ol/10(6) cells) caused a rapid and dose-dependent transient increase i n [Ca2+]i in the presence of extracellular Ca2+ that remained elevated over baseline for 15 to 30 s. In the absence of extracellular Ca2+, L OOH also evoked a transient increase in [Ca2+]i of lesser magnitude wh ich immediately returned to basal (or below basal) levels. In this reg ard, the rise in intracellular Ca2+ after LOOH or vasopressin (AVP) tr eatments involved, at least in part, related intracellular pools that in each case was followed by influx of extracellular Ca2+. The intrace llular membrane sources of Ca2+ remain unidentified as common sources known to be affected by vasopressin were not directly involved. Most n otably, the LOOH evoked rise in [Ca2+]i was not associated with releas e of IP3, suggesting that the source of intracellular Ca2+ is not IP3- sensitive pools, However, pretreatment with LOOH strongly inhibited th e rise in [Ca2+]i upon subsequent addition of AVP or LOOH and the exte nt of such inhibition was dependent on the availability of free intrac ellular Ca2+ and presence of extracellular Ca2+, These findings sugges t that reuptake of Ca2+ into intracellular membrane pools is reduced i n the presence of LOOH and/or the availability of Ca2+ from agonist-se nsitive sites is inhibited by LOOH. An increase in free 20:4 levels wa s found after LOOH treatment that was only partly prevented using intr acellular Ca2+ chelators which maintained [Ca2+]i at basal levels afte r LOOH treatment. These findings suggest that LOOH induction of phosph olipid hydrolysis proceeds following small transients in [Ca2+]i that are considerably less than that evoked by agents such as AVP, approxim ating basal Ca2+ concentrations. Inhibition of LOOH-induced lipid pero xidation by vitamin E also prevented the rise in [Ca2+]i and 20:4 rele ase indicating that phospholipid hydrolysis is dependent, at least in part, on membrane lipid peroxidation. Inhibition of protein kinase C ( PKC) completely blocked LOOH-induced release of 20:4 but had little ef fect on the LOOH-induced rise in [Ca2+]i, suggesting an indirect relat ionship between LOOH-induced membrane Ca2+ signalling events, with int ervention via PKC-mediated induction of phospholipid hydrolysis. A rap id and progressive translocation of PKC to the membrane fraction was e vident after LOOH addition over the time course corresponding to the m aximal release of 20:4 which was also inhibited by vitamin E. The find ings are discussed in terms of possible mechanisms underlying the stim ulation of phospholipase(s), purportedly phospholipase A(2) (PLA(2)) a ctivity, by peroxidation of cell membrane phospholipids and enhancemen t of protein kinase activity. (C) 1995 Academic Press, Inc.