Activation of rat basophilic leukemia cells

Antigen‐induced 45 calcium influx into rat basophilic leukemia (RBL) cells was examined with emphasis on the early time domain under conditions that exclude loss of the cation during the subsequent washing step. Such preparations demonstrate a distinct, temporally separate influx peaking at 3 min, followed by a substantial efflux. This internalized 45 Ca 2+ approaches millimolar total intracellular concentration and is therefore either sequestered or becomes bound to intracellular components (proteins). The amplitude of this influx is linearly proportional to IgE‐receptor occupancy at low receptor occupancies, and is sensitive to the anti‐allergic drug cromolyn. Furthermore, the timing of both the maximal uptake and the maximal susceptibility to cromolyn correlates with the Quin‐2 signal in these cells, and the initial degranulation pattern bears some resemblance to trends in the 45 Ca 2+ uptake curve. These qualities suggest that the early peak at 2–3 min, rather than any later 45 Ca 2+ uptake, comprises the initial signalling Ca 2+ pool. Maximal apparent inhibition by cromolyn for 45 Ca 2+ uptake was about 65% and required a 10–15‐min preincubation with the cells. The inhibitory effect was limited to the peak at 3 min, suggesting that tracer incorporation beyond 5–6 min largely involves other pools or pathways, triggered by receptor aggregation, yet only indirectly related to channel activity or to the signal proper. A quantitative similarity was found between the early peak measured on intact cells and the single channel conductance measured on reconstituted planar bilayers containing the purified receptor for IgE and the purified cromolyn‐binding protein [Corcia, A. et al. (1986) EMBO J. 5 , 849–854]. This, as well as the effects of cromolyn, support the assumption that cromolyn‐binding protein is a major constituent involved in this early influx, or that influx is a principal pathway for the signaling calcium mass.