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 cells treated with linoleic acid hydroperoxide (LOOH). LOOH (0.1–0.4) μmol/106cells) caused a rapid and dose-dependent transient increase in [Ca2+]iin the presence of extracellular Ca2+that remained elevated over baseline for 15 to 30 s. In the absence of extracellular Ca2+, LOOH also evoked a transient increase in [Ca2+]iof lesser magnitude which immediately returned to basal (or below basal) levels. In this regard, the rise in intracellular Ca2+after LOOH or vasopressin (AVP) treatments involved, at least in part, related intracellular pools that in each case was followed by influx of extracellular Ca2+. The intracellular membrane sources of Ca2+remain unidentified as common sources known to be affected by vasopressin were not directly involved. Most notably, the LOOH evoked rise in [Ca2+]iwas not associated with release of IP3, suggesting that the source of intracellular Ca2+is not IP3-sensitive pools. However, pretreatment with LOOH strongly inhibited the rise in [Ca2+]iupon subsequent addition of AVP or LOOH and the extent of such inhibition was dependent on the availability of free intracellular Ca2+and presence of extracellular Ca2+. These findings suggest that reuptake of Ca2+into intracellular membrane pools is reduced in the presence of LOOH and/or the availability of Ca2+from agonist-sensitive sites is inhibited by LOOH. An increase in free 20:4 levels was found after LOOH treatment that was only partly prevented using intracellular Ca2+chela tors which maintained [Ca2+]iat basal levels after LOOH treatment. These findings suggest that LOOH induction of phospholipid hydrolysis proceeds following small transients in [Ca2+]ithat are considerably less than that evoked by agents such as AVP, approximating basal Ca2+concentrations. Inhibition of LOOH-induced lipid peroxidation by vitamin E also prevented the rise in [Ca2+]iand 20:4 release 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 effect on the LOOH-induced rise in [Ca2+]i, suggesting an indirect rela- tionship between LOOH-induced membrane Ca2+signalling events, with intervention via PKC-mediated induction of phospholipid hydrolysis. A ra