Abstract
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 rapid and progressive translocation of PKC to the membrane fraction was evident after LOOH addition over the time course corresponding to the maximal release of 20:4 which was also inhibited by vitamin E. The findings are discussed in terms of possible mechanisms underlying the stimulation of phospholipase(s), purportedly phospholipase A2(PLA2) activity, by peroxidation of cell membrane phospholipids and enhancement of protein kinase activity.
Published Version
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