Incubation with nigericin or 20 mM NH₄Cl does not affect exocytosis by impacting Ca²⁺ influx or calcium internal stores. (A) PC12 cells loaded with Fura-2 were used to estimate variations of the cytosolic Ca²⁺ concentration. The basal Ca²⁺ concentration in the cytosol was not affected by the presence of 20 mM NH₄Cl. 5 µM nigericin elicited a progressive increase of cytosolic Ca²⁺ (probably from internal stores because no external Ca²⁺ was present). K⁺ depolarization in the presence of 2 mM external calcium induced a marked increase of the cytosolic Ca²⁺ concentration, which was not much affected by nigericin or NH₄Cl. Curves are means from 38 or 39 cells from three to four independent experiments (for clarity, SDs that were always inferior to 10% of the means are not shown). (B) Quantification of the Fura-2 signals resulting from a 5-min-long K⁺ depolarization, without (control [ctrl]) or in the presence of 5 µM nigericin (nig) or 20 mM NH₄Cl (NH4). Means ± SEM from 38 or 39 cells from three to four different cell cultures. (C) NH₄Cl (10 and 20 mM) similarly reduced CgA-EAP release induced by KCl depolarization or by the calcium ionophore A23187 (A23187) in the presence of 2 mM external calcium. Data are means ± SEM. (D) PC12 cells expressing CgA-EAP were preincubated 15 min with 10 µM thapsigargin (Thapsi) or with 10 µM calcium ionophore A23187 in the absence of external calcium before triggering CgA-EAP release by KCl. Data were obtained from four (A23187) or three (thapsigargin) different cell cultures. Data are means ± SEM. *, P < 0.05; ***, P < 0.001.