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 K_m=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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