Correlation between cytosolic calcium concentration and degranulation in RBL-2H3 cells in the presence of various concentrations of antigen-specific IgEs.
We studied the dependence of beta-hexosaminidase release from RBL-2H3 cells on the antigen-specific IgE concentrations. The cells were sensitized with DNP-specific IgE (0.5-5000 ng/ml) or OVA-specific IgE (5-50 ng/ml) and stimulated with DNP(35)-HSA (10(-2)-100 ng/ml) or OVA (10(-1) ng/ml-10 microg/ml). It was found that the beta-hexosaminidase release increased in a dose-dependent manner with the concentration of the IgEs and antigens added to the mast-cell suspension. We also studied the correlation between the intracellular calcium concentration ([Ca(2+)]i) and degranulation in RBL-2H3 cells. The percentage of beta-hexosaminidase release from the cells was well correlated with [Ca(2+)](i) increase, and the correlation coefficient was 0.88 for DNP-specific IgE and 0.99 for OVA-specific IgE. The minimum [Ca(2+)](i) required to induce the beta-hexosaminidase release was 100 nM for DNP-specific IgE, and 70 nM for OVA-specific IgE. Therefore, the [Ca(2+)](i) monitoring system is a sensitive marker of degranulation from RBL-2H3 cells and can be used to measure even low amounts of antigen-specific IgE.
Aketani
S
S
Department of Chemistry, School of Sciences, The University of Tokyo, Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan.
Teshima
R
R
Umezawa
Y
Y
Sawada
J
J
D000818:Animals; D000941:Antigens / Q000276:immunology; D002118:Calcium / Q000378:metabolism*; D015550:Cell Degranulation; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008407:Mast Cells / Q000502:physiology*; D051379:Mice; D010047:Ovalbumin / Q000276:immunology; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000941:Antigens; D007073:Immunoglobulin E; D010047:Ovalbumin; D001619:beta-N-Acetylhexosaminidases; D002118:Calcium