Direct demonstration of increased intracellular concentration of free calcium as measured by quin-2 in stimulated rat peritoneal mast cell

Abstract

Rat mast cells, passively sensitized with monoclonal mouse IgE antibody, were stimulated with multi-valent antigen, and an increase in cytoplasmic Ca2+ was determined by using the fluorescent probe quin-2. The increase in quin-2 fluorescence reached maximum within 20 sec after the antigen challenge and then gradually declined. A substantial increase in quin-2 fluorescence was observed in the presence of EGTA, indicating that bridging of cell-bound IgE antibody molecules by antigen induced not only Ca2+ influx but also mobilization of intracellular Ca2+. Phosphatidylserine added to the medium enhanced both the antigen-induced histamine release and the increase in quin-2 fluorescence and slowed the rate at which the quin-2 signal returned to basal levels. Both the antigen-induced increase in quin-2 fluorescence and histamine release were inhibited by pretreatment of mast cells with inhibitors of methyltransferases, theophylline, or cromoglycate. It was also found that methyltransferase inhibitors and theophylline inhibited not only stimulus-dependent calcium influx but also release of bound calcium from intracellular stores. Other secretagogues, compound 48/80 (1 microgram/ml) and Ca ionophore A23187 (0.1 microM), induced a rapid increase in cytoplasmic Ca2+ in rat mast cells and subsequent histamine release. In contrast, the cocarcinogenic compound phorbol 12-myristate 13-acetate caused histamine release without increasing the quin-2 fluorescence.