E3040, a new class of anti-inflammatory drug, is a dual inhibitor of 5-lipoxygenase and thromboxane synthase (Hibi et al. 1994). The major metabolite of E3040 is a sulphate conjugate (Takenaka et al. 1995). Recently, we have reported that platelet-activating factor (PAF)-induced Cl– secretion in the rat colon is mainly mediated by a release of thromboxane A2 (Suzuki et al. 2000).
Here, we examined the effects of E3040 sulphate on enzyme activities in a cell-free system and on the thromboxane A2-mediated Cl– secretion induced by PAF in isolated rat colons. Rats were killed rapidly by stunning and cervical dislocation. Enzyme immunoassay and HPLC were performed to examine the effects of E3040 sulphate on the activities of thromboxane synthase, 5-lipoxygenase and cyclo-oxygenase. Effects of the drug on the thromboxane A2-mediated Cl– secretion were examined in isolated rat distal colon mounted in an Ussing chamber. The IC50 was obtained by curve fitting averaged data. Differences between groups were analysed by one-way ANOVA.
E3040 sulphate inhibited thromboxane synthase activity in a concentration-dependent manner (IC50 = 0.013 µM), whereas it induced little effects on 5-lipoxygenase and cyclo-oxygenase activities (IC50 > 100 µM) with the cell-free enzyme assay (n = 3). With isolated rat colonic mucosa, E3040 sulphate in a concentration-dependent manner inhibited the Cl– secretion induced by PAF (IC50 = 1.8 µM, n = 5). On the other hand, E3040 sulphate (30 µM) induced no effect on the prostaglandin E2 (0.5 µM)-and leukotriene D4 (1 µM)-induced Cl– secretion in the colon (P > 0.05, n = 5). Inhibitory effect of E3040 sulphate on the PAF-induced Cl– secretion was abolished by probenecid (100 µM, P < 0.01, n = 5), an inhibitor of organic anion transporter (OAT).
Our results suggest that E3040 sulphate is a specific thromboxane synthase inhibitor, and that the drug transported into the subepithelial cells by OAT inhibits the PAF-induced Cl– secretion by blocking the release of TXA2.