Development of a sensitive method for measurement of leukotriene production by isolated cells

Washed rat peritoneal cells (RPC) rapidly and efficiently incorporated exogenous [3H]-arachidonic acid (AA). Exposure of labeled RPC to the calcium ionophore A23187 induced production of [3H]-leukotriene C4, D4 and E4 (LTC4, LTD4 and LTE4) and [3H]-prostaglandins (PGs). The radiolabeled lipoxygenase metabolites were isolated by a combination of organic extraction, silicic acid chromatography and reverse-phase high pressure liquid chromatography (RP-HPLC). Authentic leukotrienes (LT) were used to identify the biologically-synthesized products on RP-HPLC and to calculate recovery. The endogenously generated LT were also characterized by their ability to contract guinea-pig ileum and their susceptibility to soybean lipoxygenase. A23187 induced a bell-shaped concentration-dependent release of [3H]-LT which peaked at 1 X 10(-6)M ionophore. Kinetic studies revealed that ionophore stimulated the formation of mainly [3H]-LTE4 with only transient accumulation of [3H]-LTC4 and D4. In the presence of cysteine, however, production of [3H]-LTE4 was abolished with a subsequent accumulation of [3H]-LTD4. Non-steroidal antiinflammatory drugs (NSAID) moderately enhanced A23187-induced [3H]-LT production, whereas phospholipase and lipoxygenase inhibitors inhibited. The method is sensitive, reliable, and efficient for measurement of small quantities of LT produced from isolated cells.