Abstract
Dynamics of degranulation was studied in horse eosinophils by patch clamp capacitance measurements. Degranulation was stimulated by intracellular application of calcium, and GTPgammaS or guanosine 5'-(beta,gamma-imido)triphosphate at different concentrations via the patch pipette. Degranulation was quantified by measuring the delay time between the beginning of intracellular perfusion and the first exocytotic event, determining the distribution of time intervals between fusion events and the capacitance step size distributions under the different conditions. The degranulation dynamics could be well reproduced using a computer model assuming three independent rate constants for granule-plasma membrane fusion, granule fusion with already exocytosed granules, and intracellular granule-granule fusion. The rate of granule-plasma membrane fusion is sensitive to both, the GTP analog and [Ca2+]i. The rate of granule-exocytosed granule fusion is sensitive to [Ca2+]i but insensitive to the GTP analogs, and the rate of granule-to-granule fusion is sensitive to the GTP analog but insensitive to [Ca2+]i. Granule fusions with the three different target compartments thus involve different regulatory mechanisms.
Highlights
Ules are able to perform homotypic fusion among themselves
We demonstrate that the three different types of fusion events occurring during eosinophil degranulation, namely granule-plasma membrane fusion, and granule-granule fusion and granule-fused granule fusion, are differentially controlled by intracellular second messengers and that granule-granule fusion is selectively stimulated by extracellular stimulation with concanavalin A (ConA)
Using simultaneous fluorescence imaging and whole-cell membrane capacitance measurements and electron microscopy we have shown that in eosinophils focal release of granular contents is achieved by intracellular granule-granule fusion followed by compound exocytosis and by cumulative fusion of granules with the membrane of a granule that had already fused with the plasma membrane [35]
Summary
Cell Preparation—Horse eosinophils were isolated from fresh blood from the jugular vein of horses and purified over discontinuous Percoll gradients as described [10]. Isolated cells were suspended in Medium 199 containing 4 mM glutamine, 4.2 mM NaHCO3, and penicillin/streptomycin, pH 7.3–7.4, stored at room temperature, and used within 2– 4 days. After a few minutes to allow the cells to settle on the glass, the dish was perfused with standard external saline (140 mM NaCl, 5 mM KCl, 2 CaCl2, 1 mM MgCl2, 10 –20 mM glucose, 10 mM HEPES/NaOH, pH 7.2–7.3). The internal solution contained 125 mM potassium-L-glutamate, 10 mM NaCl, 7 mM MgCl2, 1 mM Na2ATP, and 10 mM HEPES/NaOH, pH 7.2–7.3. For experiments at high intracellular calcium concentration ([Ca2ϩ]i), the pipette solutions contained 5 mM EGTA and 4.5 mM CaCl2, the resulting [Ca2ϩ]i was calculated to be 1.5 M. In the solutions with low [Ca2ϩ]i (Ͻ10 nM) 7 mM EGTA was added and CaCl2 was omitted.
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