Importance of bicarbonate ion for intracellular pH regulation in antigen‐ and ionomycin‐stimulated RBL‐2H3 mast cells

In RBL-2H3 rat basophilic leukemia cells, Ca2+ influx and secretion are activated by antigens that crosslink IgEreceptor complexes and by the Ca2+ ionophore, ionomycin. Here we report that antigen-stimulated Ca2+ influx and secretion are impaired and ionomycin-induced responses are strongly inhibited following the removal of HCO3− from the medium. These results raised the possibility that HCO3− -dependent pH regulation mechanisms play a role in the cascade of events leading to mast cell activation. To test this hypothesis, intracellular pH (pHi) was measured by ratio imaging microscopy in individual RBL-2H3 cells labeled with 2',7'-bis(2-carboxyethyl)-5-(6) carboxyfluorescein (BCECF). In unstimulated cells, it was found that basal pHi in the presence of HCO3− is 7.26, significantly greater than pHi in its absence, 7.09 (P < 10−6). These results, as well as evidence that pHi increases rapidly when HCO3− is added to cells initially incubated in HCO3− -free medium, indicate that unstimulated cells use a HCO3− -dependent mechanism to maintain cytoplasmic pH. Further analyses comparing unstimulated with stimulated cells showed that antigen causes a small transient acidification in medium containing HCO3− and a larger sustained acidification in HCO3− -depleted medium. Ionomycin is a more potent acidifying agent, stimulating a sustained acidification in complete medium and causing further acidification in HCO3− -free medium. These results support the hypothesis that the inhibition of antigen- and ionomycininduced 45Ca2+ influx and secretion in cells incubated in HCO3− -free medium is at least partially due to the inactivation of HCO3− -dependent mechanisms required to maintain pH in unstimulated cells and to permit pH recovery from stimulus-induced acidification.