IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+.

ABSTRACT

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.