GPN does not release lysosomal Ca 2+ but evokes Ca 2+ release from the ER by increasing the cytosolic pH independently of cathepsin C.

The dipeptide glycyl-L-phenylalanine 2-naphthylamide (GPN) 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 (pH(ly)), and transient increases in cytosolic pH (pH(cyt)) 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 endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+](c) by increasing pH(cyt), which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pH(cyt) stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.