A transient acidification linked to intracellular Ca2 in anti-μ-stimulated human B-lymphocytes

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Abstract
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-carboxyfluorescein, 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. Our data therefore suggest that the anti-.mu.-induced rapid acidification is the result of increased intracellular Ca2+ and dependent on it.