Cells release
ATP in response to physiologic stimuli. Extracellular
ATP regulates a broad range of important cellular functions by activation of the
purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular
ATP release.
FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure
ATP release in HTC rat
hepatoma and Mz-Cha-1 human
cholangiocarcinoma cells. Exposure to a Cl(-) channel inhibitor
5-nitro-2-(3-phenylpropylamino)benzoic acid (
NPPB) (50-300 microM) stimulated a 5-100-fold increase in extracellular
ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl(-) channel activity.
NPPB also potently stimulated
ATP release in HEK293 cells and HEK293 cells expressing a rat
P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of
ATP release by
NPPB. In all cells studied,
NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of
NPPB-evoked exocytosis and
ATP release were similar. Furthermore, the magnitudes of
NPPB-evoked exocytosis and
ATP release were correlated (correlation coefficient 0.77), indicating that
NPPB may stimulate exocytosis of a pool of
ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular
ATP release and suggest that
NPPB can be used as a biochemical tool to specifically stimulate
ATP release through exocytic mechanisms.