The Lysosomotrope GPN Mobilises Ca from Acidic Organelles

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