Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes.

Elucidating mechanisms by which Ca(2+) signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca(2+) signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-β-napthylamide (GPN). Exposure to 0.2 mM GPN resulted in robust increases in the intracellular Ca(2+) concentration (Ca(2+)) in the absence of extracellular Ca(2+). The response was antagonised by thapsigargin and evoked capacitative Ca(2+) entry. Dantrolene-sensitive Ca(2+) responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca(2+) responses and β-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of Ca(2+) and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca(2+) signalling. Furthermore, the ability of GPN to raise Ca(2+) was inhibited by P2Y receptor antagonists or by inhibiting vesicular exocytosis with N-ethylmaleimide (NEM). NEM treatment was associated with an inability of GPN to trigger ATP secretion, a drop in baseline Ca(2+) and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline Ca(2+). In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca(2+) signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca(2+) homeostasis in monocytes. Lysosomal Ca(2+) stores can communicate with ER Ca(2+) stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.