GPN does not release lysosomal Ca2+, but evokes ER Ca2+ release by increasing cytosolic pH independent of cathepsin C

GPN (glycyl-l-phenylalanine 2-naphthylamide) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme, cathepsin C, is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.