title: Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes.
abstract: 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+)](i)) 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+)](i) and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca(2+) signalling. Furthermore, the ability of GPN to raise [Ca(2+)](i) 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+)](i) and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca(2+)](i). 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.
authors: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ,  UK.
authors: Venketesh
authors: V
authors: Sivaramakrishnan
authors: Stefan
authors: S
authors: Bidula
authors: Hinnah
authors: H
authors: Campwala
authors: Divya
authors: D
authors: Katikaneni
authors: Samuel J
authors: SJ
authors: Fountain
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D058470:Receptors, Purinergic P2Y / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D004151:Dipeptides; D058470:Receptors, Purinergic P2Y; C041865:glycylphenylalanine 2-naphthylamide; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium