Biphasic Ca²⁺ Responses in Human Basophils Evidence that the Initial Transient Elevation Associated with the Mobilization of Intracellular Calcium Is an Insufficient Signal for Degranulation

Human basophils exhibited biphasic cytosolic Ca²⁺ responses after stimulation with both the bacterial peptide, FMLP and IgE cross-linking agents such as goat polyclonal anti-IgE PS myeloma (anti-IgE) antibody and Ag. The biphasic response to anti-IgE antibody was not apparent unless supraoptimal concentrations of anti-IgE antibody were used to stimulate the cells. Biphasic response characteristics were also observed in single cells. As in many cell types, the first phase of the Ca²⁺ response appeared independent of extracellular Ca²⁺. Challenging basophils with anti-IgE in the presence of EGTA, added simultaneously or 3 to 5 min before the stimulus, had little effect on the first transient Ca²⁺ elevation while completely eliminating the second phase of the response. We noted that while simultaneous addition of EGTA had little effect on the histamine release after challenge with FMLP, there was no histamine release after the simultaneous addition of anti-IgE and EGTA. An examination of the kinetics of histamine release after anti-IgE also demonstrated that no histamine release occurs during the initial transient response. Since the initial Ca²⁺ transient was intact after both stimuli in the presence of EGTA, we concluded that the initial transient was not sufficient to induce histamine release. The hypothesis that distinct sources of calcium were responsible for the initial Ca²⁺ transient that followed challenge with FMLP peptide or anti-IgE antibody was also tested. We found that prior treatment of basophils with thapsigargin inhibited the initial Ca²⁺ response that followed both FMLP and anti-IgE antibody. Furthermore, stimulation with FMLP, in the presence of EGTA, depleted the calcium source responsible for the anti-IgE response. The experiment conducted in reverse order showed that anti-IgE antibody could discharge the calcium source responsible for the initial FMLP Ca²⁺ response. These experiments indicated that FMLP and anti-IgE antibody use the same internal source of calcium to produce the initial Ca²⁺ response. Taken together with previous studies on FMLP, anaphylatoxin split product of the C component, C5; and platelet activating factor-induced release in human basophils, these results suggest that the initial transient elevation, which is relatively independent of extracellular Ca²⁺, is not sufficient to induce degranulation.