Treatments that dissipate the granular pH gradient affect exocytotic release of CgA-EAP from PC12 cells. (A) PC12 cells stably expressing CgA fused with EAP (CgA-EAP) were used to estimate exocytosis of secretory granules induced by KCl depolarization (control [ctrl]). Preincubation with 20 mM NH₄Cl for 15 min decreased CgA-EAP release by 52% (NH4), and this was reversed by NH₄Cl washout (w). Washing performed in the presence of concanamycin A (w – C) led to partial reversion. (B) Dose dependence of the effect of NH₄Cl on CgA-EAP release. Release was 64.2 ± 3.1 (n = 10), 55.2 ± 2.5 (n = 13), and 44.4 ± 2.2% (n = 19) of control release at 5, 10, and 20 mM NH₄Cl, respectively. (C) A 15-min preincubation with 5 µM nigericin (nig) markedly reduced CgA-EAP release: 24.4 ± 1.2% (n = 19) of control release. This effect was not reversed after removal of nigericin (washing), 20.8 ± 1.5% (n = 11), unless the K⁺ ionophore valinomycin (5 µM) was present (washing + valinomycin [V]), a partial reversion that was inhibited by 0.4 µM concanamycin A (washing + valinomycin and concanamycin A [VC]). (D) Effects of 0.4 µM V-ATPase inhibitors. Concanamycin A (C) or bafilomycin A1 (B) did not affect CgA-EAP release, with 99.8 ± 2.5 (n = 25) and 101.1 ± 3.1% (n = 21) of control release, respectively. Saliphenylhalamide A (S) slightly but significantly depressed CgA-EAP release, with 78.5 ± 6.5% (n = 12) of control release. (E) Correlation of CgA-EAP release and intragranular pH in the presence of vehicle (1); reversion of 20 mM NH₄Cl (2); 5, 10, and 20 mM NH₄Cl (3, 4, 5, respectively); nigericin (6); bafilomycin A1 (B); and saliphenylhalamide A (S). Data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.