Mechanobiology June 26th - June 2nd 2016

Mechanobiology: June 26th  - June 2nd 2016

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes
Venketesh Sivaramakrishnan, Stefan Bidula, Hinnah Campwala, Divya Katikaneni, Samuel J. Fountain


Elucidating mechanisms by which Ca2+ signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca2+ 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 Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+. The response was antagonised by thapsigargin and evoked capacitative Ca2+ entry. Dantrolene-sensitive Ca2+ responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca2+ responses and β-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of [Ca2+]i and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca2+ signalling. Furthermore, the ability of GPN to raise [Ca2+]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 [Ca2+]i and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca2+]i. In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca2+ signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca2+ homeostasis in monocytes. Lysosomal Ca2+ stores can communicate with ER Ca2+ stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.


  • Accepted June 1, 2012.
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