The lysosomotrope GPN mobilises Ca²⁺ from acidic organelles

Lysosomes are acidic Ca²⁺ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca²⁺ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca²⁺ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca²⁺ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca²⁺ signals. NH₄Cl evoked comparable changes in pH but failed to affect Ca²⁺ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca²⁺ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca²⁺ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca²⁺ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca²⁺ release. We conclude that Ca²⁺ signals evoked by GPN most likely derive from acidic organelles.