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 multivalent antigen and an increse in cytoplasmic Ca2+ was determined by using the fluorescent probe quin-2. The increase in quin-2 fluorescence reached maximum within 20 s after the antigen challenge and then gradually declined. A substantial increase in quin-2 fluorescence was observed in the presence of EGTA [ethylene glycol-bis-[.beta.-aminoethyl ether],N,N''-tetraacetic acid], 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 retuned 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. Methyltransferase inhibitors and theophylline inhibited not only stimulus-dependent Ca influx but also release of bound Ca from intracellular stores. Other secretagogues, compound 48/80 (1 .mu.g/ml) and Ca ionophore A23187 [calcimycin] (0.1 .mu.M), induced a rapid increase in cytoplasmic Ca2+ in rat mast cells and subsequent histamine release. The cocarcinogenic compound phorbol 12-myristate 13-acetate caused histamine release without increasing the quin-2 fluorescence.