Arachidonate released upon agonist stimulation preferentially originates from arachidonate most recently incorporated into nuclear membrane phospholipids

When icosanoid-producing cells are stimulated by an agonist, 2-10% of total cellular arachidonate is released from phospholipids, and a variable percentage of the released arachidonate is subsequently converted into icosanoids. We used a mouse fibrosarcoma cell line (HSDM1C1) which synthesizes prostaglandin E2 in response to bradykinin stimulation to address the following questions: 1) upon cell stimulation is newly incorporated arachidonate preferentially released from phospholipids over previously incorporated arachidonate and 2) is there a corresponding change in phospholipid or membrane compartmentation of arachidonate to explain preferential release of newly incorporated arachidonate? To study changes in the availability of arachidonate for release from phospholipids, we incubated HSDM1C1 cells with 0.67 microM 14C]arachidonate for 15 min and chased the pulse of radiolabeled arachidonate with normal serum fatty acids. We found that of the 14C]arachidonate incorporated into phospholipids during the 15-min pulse, the percent released upon stimulation decreased nearly 3-fold from 8.9 +/- 0.5% at 5 min of chase to 3.6 +/- 0.2% (mean +/- S.E., n = 6, P less than 0.001) after only 60 min of chase. Percent release of arachidonate from nonpulsed controls was 3-4%. Although arachidonate release from phospholipids decreased significantly after 60 min of chase, the arachidonate which was released always originated predominantly from phosphatidylinositol. There was no decrease in the activities of enzymes required for arachidonate release during this time period. We also observed that throughout the period of the chase, the radiolabeled arachidonate remained esterified to the same phospholipid class into which it was initially incorporated (approximately 40% of 14C]arachidonate in diacyl phosphatidylcholine, 40% in phosphatidylinositol, and 15% in diacyl phosphatidylethanolamine. In cell fractionation experiments, we found that after 1-3 h of chase, 14C]arachidonate decreased in subcellular fractions containing nuclei, as it became progressively unavailable for release from phospholipids. Thus, our results indicate that 1) upon cell stimulation, the most recently incorporated pool of arachidonate, which is in high concentration in the nuclear membrane, is preferentially released and that 2) arachidonate rapidly moves out of the nuclear membrane into a less releasable pool while remaining esterified to the phospholipid moiety into which it was initially incorporated. This study indicates that the subcellular compartmentation of arachidonate has a marked influence on the cellular metabolism of arachidonate.