ROLES FOR CA2+ STORES RELEASE AND 2 CA2+ INFLUX PATHWAYS IN THE FC-EPSILON-R1-ACTIVATED CA2+ RESPONSES OF RBL-2H3 MAST-CELLS

Cross-linking the high affinity IgE receptor, Fc epsilon R1, with multivalent antigen induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3]-dependent release of intracellular Ca2+ stores, Ca2+ influx, and secretion of inflammatory mediators from RBL-2H3 mast cells. Here, fluorescence ratio imaging microscopy was used to characterize the antigen-induced Ca2+ responses of single fura-2-loaded RBL-2H3 cells in the presence and absence of extracellular Ca2+ (Ca-0(2+)). As antigen concentration increases toward the optimum for secretion, more cells show a Ca2+ spike or an abrupt increase in [Ca2+](i) and the lag time to onset of the response decreases both in the presence and the absence of Ca-0(2+). When Ca-0(2+) is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca-0(2+), indicating that Ca-0(2+) contributes to Ca2+ stores release. Ins(1,4,5)P-3 production is not impaired bp the removal of Ca-0(2+), suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P-3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca-0(2+), to a small, gradual increase in [Ca2+](i) before the abrupt spike response that indicates stores release. We propose that this small, initial [Ca2+](i) increase is due to receptor-activated Ca2+ influx that precedes and may facilitate Ca2+ stores release. A mechanism for capacitative Ca2+ entry also exists in RBL-2H3 cells. Our data suggest that a previously undescribed response to Fc epsilon R1 cross-linking, inhibition of Ca2+ stores refilling, may be involved in activating capacitative Ca2+ entry in antigen-stimulated RBL-2H3 cells, thus providing the elevated [Ca2+](i) required for optimal secretion. The existence of both capacitative entry and Ca2+ influx that can precede Ca2+ release from intracellular stores suggests that at least two mechanisms of stimulated Ca2+ influx are present in RBL-2H3 cells.