BW-A4C

The aim of the present study was to find out the mechanism by which the inflammatory mediator, bradykinin, induces an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in enteric neurons. For this purpose, ganglia in the isolated submucosa from rat colon were loaded with the Ca(2+)-sensitive dye, fura-2, and were exposed to bradykinin (2·10(-8)mol/l). Under control conditions, the kinin evoked a transient increase in [Ca(2+)](i). Preincubation with quinacrine or arachidonyltrifluoromethylketone (AACOCF(3)), i.e. blockers of cytosolic phospholipase A(2), prevented the raise of [Ca(2+)](i). This inhibition was mimicked by 5,8,11,14-eicosatetrayonic acid (ETYA), an inhibitor of cyclooxygenases as well as lipoxygenases, and by BWA4C, a selective inhibitor of lipoxygenases, whereas indomethacin was ineffective, suggesting the mediation of the kinin response by a lipoxygenase metabolite. Indeed, a leukotriene, leukotriene D(4) (LTD(4)), mimicked the effect of bradykinin. The LTD(4) receptor blocker, MK-571, inhibited the increase in [Ca(2+)](i) evoked by LTD(4) and by bradykinin. Consequently, bradykinin receptors in submucosal ganglia from rat colon are coupled to a stimulation of phospholipase A(2), the release of arachidonic acid and the production of LTD(4), which seems to be finally responsible for the change in the cytosolic Ca(2+) concentration.

BACKGROUND AND PURPOSE 5‐Lipoxygenase (5‐LO) is the key enzyme in the biosynthesis of pro‐inflammatory leukotrienes (LTs) representing a potential target for pharmacological intervention with inflammation and allergic disorders. Although many LT synthesis inhibitors are effective in simple in vitro test systems, they frequently fail in vivo due to lack of efficacy. Here, we attempted to assess the pharmacological potential of the previously identified 5‐LO inhibitor 2‐(4‐(biphenyl‐4‐ylamino)‐6‐chloropyrimidin‐2‐ylthio)octanoic acid (HZ52).EXPERIMENTAL APPROACH We evaluated the efficacy of HZ52 in vivo using carrageenan‐induced pleurisy in rats and platelet‐activating factor (PAF)‐induced lethal shock in mice. We also characterized 5‐LO inhibition by HZ52 at the cellular and molecular level in comparison with other types of 5‐LO inhibitor, that is, BWA4C, ZM230487 and hyperforin.KEY RESULTS HZ52, 1.5 mg·kg−1 i.p., prevented carrageenan‐induced pleurisy accompanied by reduced LTB4 levels and protected mice (10 mg·kg−1, i.p.) against PAF‐induced shock. Detailed analysis in cell‐based and cell‐free assays revealed that inhibition of 5‐LO by HZ52 (i) does not depend on radical scavenging properties and is reversible; (ii) is not impaired by an increased peroxide tone or by elevated substrate concentrations; and (iii) is little affected by the cell stimulus or by phospholipids, glycerides, membranes or Ca2+.CONCLUSIONS AND IMPLICATIONS HZ52 is a promising new type of 5‐LO inhibitor with efficacy in vivo and with a favourable pharmacological profile. It possesses a unique 5‐LO inhibitory mechanism different from classical 5‐LO inhibitors and seemingly lacks the typical disadvantages of former classes of LT synthesis blockers.