Rabbits were imn~unized with a conjugate of leukotriene (LT) C4 and bovine serum albumin prepared by coupling the single free amino group of the hapten to the protein using gluteraldehyde. Binding of [3H]LTC4 to the antibodies obtained is inhibited by 50% with 1.5 ng LTC4. The relative cross-reaction of LTD 4 is 16% and of LTC4-methyl ester 3.6%. The validity of the radioimmunoassay was demonstrated by comparison with bioassay using the isolated guinea pig ileum. Using the radioimmunoassay it could be shown that endogenous LTC4 is released in a dose-dependent manner by human polymorphonuclear leucocytes stimulated with the divalent cation ionophore A23187. Leukotriene C4 Radioimmunoassay Leucocyte Bioassay LipoxygenaseSlow-reacting substance

Leukotriene and prostaglandin production by mouse peritoneal macrophages was investigated. It could be shown that the tumour promoter 12-O-tetradecanoylphorbol-13-acetate initiated the release of prostaglandin E2 but had little effect on the release of leukotriene C4-like immunoreactivity. The divalent cation ionophore A 23187 at concentrations between 10(-6) and 10(-8) mol/l initiated prostaglandin as well as leukotriene release. This prostaglandin and leukotriene release could be modulated by drugs. Non-steroidal anti-inflammatory drugs including benoxaprofen inhibited prostaglandin release but simultaneously enhanced leukotriene production. The analgesics paracetamol and 4-methylaminoantipyrine had similar effects at high concentrations. The experimental compound BW 755 c inhibited prostaglandin and leukotriene production while the antithrombotic compound nafazatrom inhibited the production of leukotriene C4-like immunoreactivity but enhanced prostaglandin E2 production. Nordihydroguaiaretic acid inhibited prostaglandin and leukotriene production. The results show that the metabolism of arachidonic acid in macrophages via the cyclooxygenase or the lipoxygenase pathway is dependent on the stimulus applied. Both pathways can be inhibited conjointly or selectively by drugs. Our results do not provide evidence that differences in anti-inflammatory activity claimed for some of the drugs tested can be explained by differential inhibition of either pathway. The experimental system described may be used for assessing the potency of drugs to inhibit the lipoxygenase and the cyclooxygenase pathway of arachidonic acid metabolism.

1 The effects of infusions of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 1.1 x 10-molmin-') and the antagonist of slow-reacting substance of anaphylaxis (SRS-A) FPL 55712 (1.2 x 10-mol min-') on the coronary constriction and the release of SRS-A, leukotriene C4-like immunoreactivity, thromboxane B2 and 6-keto-prostaglandin Fl. from perfused anaphylactic guinea-pig hearts were investigated. 2 Both NDGA and FPL 55712 in the concentrations used induced an increase in basal coronary flow, but did not prevent the coronary flow reduction in the early phase (0-4 min) after antigen injection. On the other hand, NDGA and FPL 55712 inhibited the less pronounced long-lasting coronary flow reduction in the later phase of cardiac anaphylaxis. 3 NDGA decreased the release of SRS-A from the anaphylactic guinea-pig hearts below or close to the detection limit of the bioassay and simultaneously diminished the release of leukotriene C4-like immunoreactivity. On the other hand, FPL 55712 did not influence the amounts of leukotriene C4-like immunoreactivity released in cardiac anaphylaxis. 4 Neither NDGA nor FPL 55712 affected the release of immunoreactive thromboxane B2 (TXB2) from anaphylactic guinea-pig hearts. Release of 6-keto-prostaglandin Flm after challenge, however, was decreased by NDGA, while FPL 55712 had no significant effect. 5 These results suggest, that SRS-A may be a relatively more important mediator in the late phase of coronary constriction occurring during cardiac anaphylaxis, while the effects of other mediators, particularly vasoconstrictor cyclo-oxygenase products, seem to prevail in the early phase.

It is known that both vasoconstrictor cyclooxygenase products and sulfidopeptide-containing leukotrienes (LT) contribute to the biphasic coronary constriction observed in isolated perfused anaphylactic guinea-pig hearts. We have now investigated the effects of the cyclooxygenase inhibitor indomethacin and of several exogenous prostaglandins (PG) on the release of LTC4-like immunoreactivity and on various symptoms of cardiac anaphylaxis. Indomethacin decreased basal coronary flow and delayed the onset of coronary vasoconstriction after antigenic challenge. Furthermore, indomethacin inhibited cardiac release of 6-keto-PGF1 alpha and thromboxane (TX) B2 and simultaneously enhanced the antigen-induced release of LTC4-like immunoreactivity significantly. Neither the vasodilators PGE2 and PGI2 nor the vasoconstrictors PGF2 alpha, PGD2 and 11,9-epoxymethano-PGH2, a compound with biological properties similar to TXA2, affected the anaphylactic release of immunoreactive LTC4 in the presence of indomethacin. These results suggest that the indomethacin-induced increase in LT release is not due to inhibition of synthesis of a cyclooxygenase product, which normally curbs anaphylactic release of immunoreactive LTC4. The indomethacin effect may rather be explained by diversion of arachidonic acid metabolism away from fatty acid cyclooxygenase towards the synthesis of lipoxygenase products. Although the various PG did not significantly affect cardiac release of LTC4-like immunoreactivity, they antagonized the anaphylactic coronary constriction. This antagonism may be due to direct effects of the PG on vascular smooth muscle tone as well as to indirect effects on the release of anaphylactic mediators not related to LT like histamine and platelet-activating factor.(ABSTRACT TRUNCATED AT 250 WORDS)

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