Contributing factors in the trafficking of [3H]arachidonate between phospholipids

Cultured human promyelocytic leukemia cells (HL-60), depleted of arachidonic acid by continued growth in serum-free media, were used as a model system to examine various factors that control the incorporation and distribution of [3H]arachidonic acid into classes and subclasses of cellular lipids. Increasing the culture media concentration of [3H]arachidonic acid from 1 x 10(-8) M to 1 x 10(-5) M caused a greater percentage of the cellular tritium to be distributed into triacylglycerols (from less than 1% at 1 x 10(-8) M to 38% at 1 x 10(-5) M) with a corresponding decrease in cellular [3H]diradylglycerophosphoethanolamine (from 53% at 1 x 10(-8) M to 12% at 1 x 10(-5) M) during 2 h incubations. A greater proportion of the tritium present in diradylglycerophosphoethanolamine and diradylglycerophosphocholine, at the higher media concentration of [3H]arachidonic acid (1 x 10(-5) M), was found in the diacyl subclasses of these two lipids than was observed at the lower concentrations (less than 1 x 10(-6) M) of [3H]arachidonic acid. Significant amounts of diarachidonoyl molecular species were found in the phosphatidylethanolamine (10%) and phosphatidylcholine (15%) of HL-60 cells that were labeled for 2 h with 1 x 10(-5) M [3H]arachidonic acid. This was the only molecular species of phosphatidylcholine to completely disappear when prelabeled cells were placed in arachidonate-free media for 22 h. Prelabeling-chase experiments with 1 x 10(-5) M [3H]arachidonic acid were consistent with movement of [3H]arachidonate from triacylglycerols into diradylglycerophosphatides and from diacylphospholipids into ether-linked phospholipids. Increasing the concentration of HL-60 cells in the incubations influenced the distribution of [3H]arachidonic acid in cellular lipid classes in a manner analogous to decreasing the concentration of [3H]arachidonic acid in the media. Increasing the endogenous level of cellular arachidonate in phospholipid classes with supplements of unlabeled arachidonic acid changed the subsequent lipid class distribution of a low concentration (1 x 10(-8) M) of [3H]arachidonic acid to resemble results obtained with a much higher mass level of [3H]arachidonate in arachidonate depleted cells. HL-60 cells differentiated into granulocytes by treatment with dimethyl sulfoxide incorporated less [3H]arachidonic acid but had a greater proportion associated with alkylacylglycerophosphocholine and alk-1-enylacylglycerophosphoethanolamine than undifferentiated HL-60 cells.