Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation

We have studied the capacity of human neutrophils to release arachidonic acid from diacylglycerol, employing 1-stearoyl-2-1-14C]arachidonoyl-sn-glycerol and 1-1-14C]stearoyl-2-arachidonoyl-sn-glycerol as exogenous substrates. We have found that arachidonic acid is removed from diacylglycerol by the sequential action of two enzymes. First, the sn-1 position is split by 1-diacylglycerol lipase activity, and then, arachidonic acid is released from the resulting 2-monoacylglycerol by a 2-monoacylglycerol lipase. The specific activity of the 2-monoacylglycerol lipase, using 2-1-14C]arachidonoyl-sn-glycerol as exogenous substrate, was at least 9-fold higher than that of 1-diacylglycerol lipase, indicating that the action of the 1-diacylglycerol lipase is the rate-limiting step in arachidonic acid release from diacylglycerol. Postnuclear supernatants from A23187-treated cells showed a 2.5-fold increase in both lipase activities. The arachidonic acid-releasing diacylglycerol lipase system showed an optimum pH of 4.5 and was not inhibited by EGTA or stimulated by Ca2+, Mg2+, Mn2+, Zn2+, or Co2+. However, arachidonic acid release was inhibited by Hg2+, suggesting the involvement of sulfhydryl groups in catalytic activity. The subcellular distribution of both 1-diacylglycerol lipase and 2-monoacylglycerol lipase activities was examined in resting and A23187-treated human neutrophils by fractionation of postnuclear supernatants on continuous sucrose gradients. Both lipases were localized mainly in the membrane of gelatinase-containing granules, which were resolved from cytosol, plasma membrane, phosphasomes, and specific and azurophilic granules. When neutrophils were stimulated by the calcium ionophore A23187, a drastic shift of the 1-diacylglycerol lipase and 2-monoacylglycerol lipase toward the plasma membrane was detected. This shift was due to fusion of gelatinase-containing granules with the plasma membrane upon neutrophil stimulation. As a result of the membrane fusion process, the capacity to release arachidonic acid from diacylglycerol was increased. This translocation from the membrane of gelatinase-containing granules to the plasma membrane may play an important role in regulating the diacylglycerol level in stimulated human neutrophils.