Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

Abstract

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B 4 (LTB 4) and thromboxane B 2 (TXB 2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B 4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC 50 1.6 × 10 −4M) was approximately 100 times less potent than BW755C (IC 50 1.7 × 10 −6M) at inhibiting leukotriene B 4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10 −6M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.