Quantitative and realtime correlation between receptor aggregation and intracellular calcium signal transduction

Quantitative correlation between intracellular calcium signals and hapten density or molecular size of antigens was studied for two cell lines; hapten-specific murine B cells (TP67.21) and rat basophilic leukemia cells (RBL-2H3) with hapten-specific IgE. Magnitude of the induced calcium signal in both cells exhibited the same dependence on hapten density of antigen molecules and there existed an optimal hapten density which induced the maximum amount of calcium signal for both cells. However, they responded differently to antigens of various molecular size. In contrast to TP67.21 cells which showed larger response to larger antigen molecules, RBL-2H3 cells showed the largest response to the smallest antigen. This may possibly suggest that there exists an optimal structure of receptor aggregates for each cell. Calcium signal induced in each cell by multivalent antigen was rapidly abrogated by addition of excess hapten and this abrogation occurred both in transmembrane influx and the release from intracellular stores. We directly observed the mobilization of receptor molecules during this calcium signal abrogation at single cell level by using two fluorescent calcium probes, whose fluorescence wavelength ranges have least overlap, and confocal microscopy. During this abrogation, large clusters of receptor molecules were not affected by hapten molecules. We, therefore, conclude that these large clusters are inactive in the induction of calcium signal and smaller clusters of receptor molecules are necessary for calcium signal induction.