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+.
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.
Hohman
R J
RJ
Allergic Diseases Section, National Institutes of Health, Bethesda, MD 20892.
Hultsch
T
T
Talbot
E
E
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D015550:Cell Degranulation; D005089:Exocytosis; D006868:Hydrolysis; D007038:Hypotonic Solutions / Q000494:pharmacology; D015471:Leukemia, Basophilic, Acute / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D011961:Receptors, Fc / Q000502:physiology*; D017455:Receptors, IgE; D014407:Tumor Cells, Cultured
D000944:Antigens, Differentiation, B-Lymphocyte; D007038:Hypotonic Solutions; D010716:Phosphatidylinositols; D011961:Receptors, Fc; D017455:Receptors, IgE; D011188:Potassium; D002118:Calcium