Role of arachidonic acid in the biosynthesis of slow reacting substance of anaphylaxis (SRS-A) from sensitized guinea pig lung fragments: evidence that SRS-A is very similar or identical structurally to nonimmunologically induced forms of SRS.

Abstract The possible precursor role of arachidonic acid (AA) in the generation of slow reacting substance of anaphylaxis (SRS-A) in antigen-stimulated guinea pig lung fragments was studied by using an approach similar to that described previously by this laboratory for nonimmunologically induced SRS production in rat basophilic leukemia (RBL-1) cells (Proc. Natl. Acad. Sci. 74:4577, 1977). 5,8,11,14-eicosatetraynoic acid (ETYA), an acetylenic analog of AA that inhibits both the lipoxygenase and cyclooxygenase pathways, substantially decreased SRS-A generation in lung at low jug/ml concentrations. On the other hand, indomethacin (IM), a selective inhibitor of the cyclooxygenase pathway, significantly enhanced SRS-A formation. Low (3 to 6 µg/ml) concentrations of AA were modestly stimulatory. The question of whether the SRS-A produced by sensitized guinea pig lung incorporates radiolabeled [14C]-AA into its structure also was investigated. Lung fragments were stimulated with antigen for 20 min in the presence of [14C]-AA. The SRS-A was purified by using a series of chromatographic and organic solvent extraction procedures including 80% ethanol extraction, adsorption chromatography on Sephadex LH-20, extraction with organic solvent at acid pH, ion exchange chromatography on DEAE-Sephadex A-25, and partition chromatography on Sephadex LH-20. The overall recovery of smooth muscle contracting activity with this purification procedure was 45%. One of the peaks of radioactivity from the final partition chromatography purification step corresponded to that of SRS-A spasmogenic activity. The radioactivity present in this fraction represented 0.03% of the [14C]-AA radioactivity originally added to the lung fragments. The very close correspondence of spasmogenic activity and radioactivity was confirmed by high pressure liquid chromatography (HPLC). The SRS-A was indistinguishable in its chromatographic behavior to the SRS produced by RBL-1 cells in response to the divalent cation ionophore A23187 (Biochem. Biophys. Res. Commun. 89: 1187, 1979). After sequential degradation of the purified SRS-A with Na in liquid ammonia and hydrazine, 5-hydroxy-arachidic acid was identified, indicating the presence of an oxygen function at the 5 position of the original SRS-A molecule. Amino acid analysis gave 0.85 mole of glycine and 0.18 mole of cysteine per mole of SRS-A, indicating that the sulfur containing side chain is cysteinyl-glycine. There was a 280 nm absorption maximum that shifted to 308 nm in the presence of soybean lipoxygenase indicating the presence of double bonds in the 7, 9, 11, and 14 positions and that the side chain is located at the 6 position. These observations indicate that AA is a metabolic precursor of SRS-A in guinea pig lungs, that a lipoxygenase acting selectively at the 5 position of AA is probably involved, and that there are marked chemical similarities between immunologically and nonimmunologically produced forms of SRS.