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 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 (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 less than 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 ionomycin-induced 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.
Lee
R J
RJ
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
Oliver
J M
JM
Deanin
G G
GG
Troup
C D
CD
Stump
R F
RF
D000818:Animals; D000941:Antigens / Q000502:physiology*; D001639:Bicarbonates / Q000032:analysis / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects; D005452:Fluoresceins; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology*; D015471:Leukemia, Basophilic, Acute / Q000473:pathology*; D008407:Mast Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D000941:Antigens; D001639:Bicarbonates; D003470:Culture Media; D005452:Fluoresceins; D015759:Ionomycin; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D002118:Calcium