IMPORTANCE OF BICARBONATE ION FOR INTRACELLULAR PH REGULATION IN ANTIGEN-STIMULATED AND IONOMYCIN-STIMULATED RBL-2H3 MAST-CELLS

In RBL-2H3 rat basophilic leukemia cells, Ca2+ influx and secretion are activated by antigens that crosslink IgE-receptor 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 (pH(i)) 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 pH(i) in the presence of HCO3- is 7.26, significantly greater than pH(i) in its absence, 7.09 (P < 10(-6)). These results, as well as evidence that pH(i) 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 ionomycin-induced Ca-452+ 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.