Roles for Ca2+ stores release and two 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)P3]-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+ (Ca2+o). 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 Ca2+o. When Ca2+o is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca2+o, indicating that Ca2+o contributes to Ca2+ stores release. Ins(1,4,5)P3 production is not impaired by the removal of Ca2+o, suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca2+o, to a small, gradual increase in [Ca2+]i before the abrupt spike response that indicates store 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.
Lee
R J
RJ
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Oliver
J M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002413:Cations, Divalent; D002460:Cell Line; D004140:Dinitrophenols / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D017136:Ion Transport / Q000187:drug effects; D008407:Mast Cells / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002413:Cations, Divalent; D004140:Dinitrophenols; D005456:Fluorescent Dyes; D017455:Receptors, IgE; D013729:Terpenes; C034638:dinitrophenyl-bovine serum albumin; C049925:fura-2-am; D012710:Serum Albumin, Bovine; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2