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Granule-specific ATP requirements for Ca²⁺-induced exocytosis in human neutrophils. Evidence for substantial ATP-independent release

¹ Division of Clinical Biochemistry, University of Geneva Medical School, 1211 Geneva 14, Switzerland
² Division of Infectious Diseases, University Hospital Geneva, 1211 Geneva 14, Switzerland
³ Immunology Laboratory, Faculty of Sciences, University Nancy 1, 54506 Vandoeuvre-les-Nancy, France

View larger version (13K): [in a new window]   Fig. 3. ATP dose-response curve. (A) Relationship between total increase in capacitance Cm (measured as the difference between the final plateau value and the initial capacitance) and time in ATP-depleting medium. The time was counted until the time of patch rupture. Open squares and filled circles represent mean values using 300 µM Ca2+ (n=3-13) and 10 µM Ca2+ (n=5-10) in the pipette, respectively. (B) The same data sets as in A are shown but plotted as a function of intracellular ATP, as calculated using the fitting function from Fig. 1B, at the time of patch rupture. Solid lines in B are logistic functions fitted to the data. For 10 µM Ca2+ Km=328 µM and the cooperativity factor P=2.0, for 300 µM Ca2+ Km=95 µM and P=3.7. (C) Reversibility of ATP depletion. Cells were ATP-depleted for 39±6.5 minutes or 65±8 minutes to lower intracellular ATP to the critical level for secondary/tertiary granules and primary granules, respectively. These cells were then stimulated with 10 µM Ca2+ or 300 µM Ca2+, respectively, including 1 mM ATP in the patch pipette (reperfusion). Mean capacitance increase from control cells (no ATP depletion) or cells reperfused with ATP after depletion, n=4-8.

View larger version (16K): [in a new window]   Fig. 1. ATP measurements with the luciferase assay. (A) Calibration curve showing that the assay is linear. (B) The calculated intracellular ATP concentrations after various times of ATP depletion. Data were pooled from four independent experiments. The line represents a sum of two exponential functions: [ATP]=2111 µMxe-t/3.5+250 µMxe-t/27. This function is used to calculate the intracellular ATP concentration at any given time of ATP depletion.

View larger version (19K): [in a new window]   Fig. 2. Ca2+-induced exocytosis after ATP depletion. Representative patch-clamp capacitance traces in control conditions, using pipette solutions containing 1 mM ATP, and after various times of incubation in an ATP-depleting medium using ATP-free pipette solutions. The incubation times until patch rupture in minutes are indicated next to the traces. In A pipette solutions contained 10 µM Ca2+ and in B 300 µM Ca2+. Time is given from the time of patch rupture. Initial capacitances have been subtracted for clarity. (C) Initial capacitance (Ci) versus time of ATP depletion with linear regression Ci(t)=0.0008 pF/minutext+2.692 pF.

View larger version (14K): [in a new window]   Fig. 4. ATP dependence of the rate of exocytosis. Relationship between the maximum rate of exocytosis, measured as the maximum value of the derivative of capacitance traces, and intracellular ATP at the time of patch rupture. A represents mean values obtained using 10 µM Ca2+ (n=5-10) and (B) using 300 µM Ca2+ (n=3-13) in the pipette. Both sets of values are normalised to their respective value in the control situation. The solid line represents logistic functions fitted to the data. In A Km=476 µM and a cooperativity factor P=2.6 was obtained. Corresponding values in B were Km=99 µM and P=7.9.

View larger version (15K): [in a new window]   Fig. 5. ATP dependence of primary granule release. (A) ATP dependence of primary granule exocytosis elicited by either 1 µM ionomycin (squares, mean of two to six experiments) or 1 µM fMLP (triangles, mean of two to four experiments). Exocytosis was measured using the ß-glucuronidase release assay. Both sets of data were normalised to their value in the control situation. The ionomycin data were fitted to a logistic function having a Km=55 µM and a cooperativity factor P=4. (B) Comparison between the ATP dependence of primary granule exocytosis as measured by the ß-glucuronidase release assay (squares, same data as in A) and as calculated from the patch-clamp measurements (solid line). The solid line was obtained as the difference between the logistic functions (in Fig. 3B) fitted to the data points obtained using 300 µM Ca2+ and 10 µM Ca2+ in the pipette. (C) Ratiometric measurements of intracellular Ca2+ concentrations after ATP depletion using fura-2/AM. The trace, an average of four experiments, is the ratio of fura-2 fluorescence at 340/380 nm excitation. Standard errors of the mean are represented by grey shading. The thick bars represent the sequential addition of 6 mM deoxyglucose, 2 µM FCCP and 1 µM ionomycin. In the calibration scaling to the right, each scale step represents an increase of 200 nM Ca2+.

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