Abstract
The esterification of endogenously formed 5-hydroxyeicosatetraenoic acid (5-HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5-HETE was observed after a 5-min stimulation of PMNL with A23187. The amount of free 5-HETE then declined rapidly, while that of 5-HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time-dependent manner. Stimulation by A23187 yielded approximately 100 ng/10(7) cells esterified 5-HETE in 60 min, which corresponded to the decrease in the amount of free 5-HETE from 5 min to 60 min and indicated that free 5-HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids. The esterification profile of exogenous 5-HETE was different from that of endogenous 5-HETE. 5-[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed that endogenous 5-HETE was esterified equally to phospholipids and triacylglycerol. Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5-HETE. However, less 5-HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12-HETE, produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation. Exogenous 5-HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12-HETE into macrophages was observed. The rapid uptake of exogenous 5-HETE was strongly inhibited by the suppression of acylation of 5-HETE by triacsin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5-HETE.
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