A transient acidification linked to intracellular Ca2+ in anti-mu-stimulated human B-lymphocytes.
Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca2+ concentration. We studied the patterns of pH and Ca2+ changes in normal resting human B-lymphocytes after exposure to anti-mu antibodies and the monoclonal antibody IF5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G1 phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-mu antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10-15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca2+ was measured by using the fluorescent Ca2+ dye, indo-I. Exposure of cells to anti-mu resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca2+ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-mu, IF5 caused no change in cytoplasmic Ca2+ and pH. However, the Ca2+ ionophore ionomycin at low concentrations mimicked the Ca2+ response as well as the pH response to anti-mu. In Ca2(+)-free solutions the intracellular Ca2+ stores are usually rapidly depleted and, indeed, the Ca2+ and pH responses to anti-mu were reduced after 5 min and almost abolished after 35 min under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Institute of Physiology, University of Oslo, Norway.
H
H
Schøyen
J G
JG
Iversen
E B
EB
Smeland
R
R
Heikkilä
D000584:Amiloride / Q000494:pharmacology*; D000888:Antibodies, Anti-Idiotypic / Q000502:physiology*; D000911:Antibodies, Monoclonal / Q000502:physiology; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology; D001402:B-Lymphocytes / Q000032:analysis* / Q000187:drug effects; D002118:Calcium / Q000032:analysis* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007148:Immunoglobulin mu-Chains / Q000276:immunology*; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D000944:Antigens, Differentiation, B-Lymphocyte; D007148:Immunoglobulin mu-Chains; D015759:Ionomycin; D000584:Amiloride; D002118:Calcium