Fig. 5.

Other weak bases evoke ER-dependent Ca²⁺ signals. (A) Structures and mechanisms of action of some lysosomotropic drugs. (B) Confocal images of HEK cells loaded with LysoTracker Red (100 nM, 20 min) and then treated (200 s) with NH₄Cl (20 mM) or fluoxetine (300 μM). (C) Quantification of fluorescence (F/F₀) from images similar to those in panel B. Results show mean±s.e.m., n=7. F₀ and F are fluorescence recorded before and 200 s after the addition of NH₄Cl or fluoxetine. (D) Effects of NH₄Cl (20 mM) or fluoxetine (300 μM) on pH_cyt recorded in populations of SNARF-5F-loaded HEK cells. Results show mean±s.d. of n=3 determinations. (E) Summary results (mean±s.e.m., n=3, each with 3 replicates) show peak increases in pH_cyt (ΔpH_cyt). (F-H) Effects of thapsigargin (TG, 1 μM, 15 min) in Ca²⁺-free HBS on the Ca²⁺ signals evoked by d-GPN (200 μM) (F), fluoxetine (300 μM) (G) or NH₄Cl (20 mM) (H). Results show mean±s.d. of n= 3 determinations. (I-K) Summary results from analyses similar to those in panels F-H show the effects of d-GPN (I), fluoxetine (J) or NH₄Cl (K) on Δ[Ca²⁺]_c alone or after treatment with thapsigargin (TG, 1 μM, 15 min). Results show mean±s.e.m., n=3, each with 3 replicates. (L) A requirement for ER Ca²⁺ for GPN to evoke an increase in [Ca²⁺]_c might reflect a need for the ER to fuel lysosomal Ca²⁺ uptake (option 1) or a direct action of GPN on the ER (option 2).