GPN does not release lysosomal Ca but evokes Ca 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), and transient increases in cytosolic pH (pH) and Ca concentration ([Ca]). 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 [Ca] require Ca within the endoplasmic reticulum (ER), but they are not mediated by ER Ca channels amplifying Ca release from lysosomes. GPN increases [Ca] by increasing pH, which then directly stimulates Ca release from the ER. We conclude that physiologically relevant increases in pH stimulate Ca release from the ER in a manner that is independent of IP and ryanodine receptors, and that GPN does not selectively target lysosomes.