Arachidonate-induced eicosanoid synthesis in RBL-2H3 cells: stimulation with antigen or A23187 induces prolonged activation of 5-lipoxygenase.

Abstract

We studied the ability of rat basophilic leukemia (RBL-2H3) cells stimulated with either IgE/antigen or calcium ionophore, A23187, to synthesize LTC4 and PGD2 after addition of exogenous arachidonic acid. RBL-2H3 cells preferentially synthesized PGD2 in response to stimulation with low concentrations of antigen or A23187 while higher concentrations also resulted in a marked synthesis of LTC4. The synthesis of LTC4 was dependent upon initial activation of 5-lipoxygenase by IgE/antigen or A23187, since arachidonic acid lone failed to induce LTC4 synthesis. Following the addition of IgE/antigen or A23187 alone, the synthesis of PGD2 and LTC4 was essentially complete by 10 min. To determine whether a limitation of substrate precluded further eicosanoid synthesis, exogenous arachidonic acid was added to washed cells 15-145 min following the initial stimulation with IgE/antigen or A23187, PGD2 and LTC4 synthesis was resumed following the addition of arachidonic acid to washed prestimulated cells, demonstrating that the termination of eicosanoid synthesis in RBL-2H3 cells was nor caused by the inactivation of cyclooxygenase and 5-lipoxygenase. DNP-lysine was added to cells previously stimulated with IgE/antigen to stop receptor aggregation and this greatly inhibited subsequent production of LTC4 following the addition of arachidonic acid, suggesting that ongoing stimulation of Fc epsilon XsRI was required for LTC4 synthesis in this setting. These results indicate that the magnitude of a physiologic stimulus (IgE/antigen) can profoundly affect the arachidonate metabolites produced by mast cells and that the synthesis of these metabolites quickly becomes limited by substrate availability rather than the activity of cyclooxygenase or 5-lipoxygenase.