Abstract
ABSTRACTLysosomes 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.
Highlights
Release of stored Ca2+ is a ubiquitous means to generate cytosolic Ca2+ signals (Clapham, 2007)
Chief among the so-called acidic Ca2+ stores are lysosomes that maintain a luminal pH of ∼4.5 and a Ca2+ concentration of ∼500 μM, similar to the endoplasmic reticulum (ER) (Christensen et al, 2002). These stores are mobilised through activation of Ca2+-permeable channels, such as two-pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs), by signalling molecules, such as nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5bisphosphate [PI(3,5)P2] (Grimm et al, 2012; Patel, 2015)
Glycyl-L-phenylalanine 2-naphthylamide (GPN) and NH4Cl differentially affect cytoplasmic Ca2+ and pH To explore the mechanism underlying GPN-evoked Ca2+ signals, we investigated the relationship between changes in Ca2+ and pH in primary cultured human fibroblasts
Summary
Release of stored Ca2+ is a ubiquitous means to generate cytosolic Ca2+ signals (Clapham, 2007). Chief among the so-called acidic Ca2+ stores are lysosomes that maintain a luminal pH of ∼4.5 and a Ca2+ concentration of ∼500 μM, similar to the ER (Christensen et al, 2002). These stores are mobilised through activation of Ca2+-permeable channels, such as two-pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs), by signalling molecules, such as nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5bisphosphate [PI(3,5)P2] (Grimm et al, 2012; Patel, 2015).
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