The release of slow reacting substance (SRS) from rat basophilic leukemia cells (RBL-1) by the ionophore A23187 (5-10 Ag/ml) was stimulated 5-fold by arachidonate and inhibited 78% by 5,8,11,14-eicosatetraynoate (an inhibitor of both fatty acid cyclooxygenase and lipoxygenase). Linoleic acid and linolenic acid both inhibited SRS formation, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Radiolabel from ["4C-or [3H]arachidonate was incorporated into SRS as indicated by comigration of radioactivity and bioreactivity in several chromatographic systems after purification to apparent radiochemical homogeneity. The radiolabeled SRS was clearly separated chromatographically from other known arachidonate metabolites. Thus, SRS appears to be a previously undescribed product of arachidonic acid metabolism, probably formed through the lipoxygenase pathway. The ability to prepare purified, biosyntletically labeled, SRS should be of considerable help in further studies of its structure, biologic function, and catabolism. Slow reacting substance (SRS) is a polar acidic lipid (or group of related lipids) of unknown structure with potent contractile activity for bronchial and ileal smooth muscle. It is readily distinguished from prostaglandins (PGs), thromboxane A2, and other agents known to contract smooth muscle by its spectrum of muscle activity (1), inhibition by the specific SRS antagonist FPL 55712 (2), and various chromatographic and physicochemical criteria (3,4). SRS is formed and released from lung, leukocytes, and other tissues during IgE-mediated allergic reactions as well as in response to a variety of nonimmunologic stimuli, the most potent of which is the calcium ionophore A23187 (1,(5)(6)(7)(8)(9). We have recently demonstrated that a line of rat basophilic leukemia cells (RBL-1) (10) with receptors for IgE (11) releases considerable quantities of SRS upon stimulation with A23187 (12, 13). The SRS from RBL-1 cells is very similar or identical in its biological and chromatographic properties to previously described SRSs from various cat, rat, or human tissues (12). With the availability of a cell line that makes large amounts of SRS, studies of SRS structure and biosynthesis should be considerably facilitated. Because SRS is an acidic lipid, and metabolites of arachidonic acid (AA) have recently been recognized as showing a wide range of biological and biochemical properties (reviewed in ref. 14), we have studied the possible role of AA in SRS formation in RBL-1 cells. Strong evidence implicating AA as a biosynthetic precursor of SRS will be described. (Sigma). A23187 was dissolved in dimethylsulfoxide and diluted with incubation medium (see below) immediately before addition to cells. Over the concentration range present in our incubation mixtures dimethylsulfoxide itself had no effect on SRS formation. None of the reagents used in this study, including AA, had significant effects in our standard guinea pig ileal bioassay system.
MATERIALS AND METHODSRBL-1 cells were grown in Eagle's m...
