Abstract
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.
Highlights
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)2 is widely used as an inhibitor of many different ClϪ channels and has been reported to inhibit ATP release mediated by ClϪ channels [16, 17]
Using FM1-43 fluorescence to measure exocytosis and bioluminescence assay to measure ATP release in real time, we found that exposure to NPPB under basal conditions potently stimulates ATP release
DMSO had no effect on the luciferin-luciferase assay or ATP release in both cells. These results indicate that NPPB stimulates ATP
Summary
5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)2 is widely used as an inhibitor of many different ClϪ channels and has been reported to inhibit ATP release mediated by ClϪ channels [16, 17]. To assess whether ATP release evoked by NPPB was caused by disruption of the plasma membrane and a leakage of ATP into extracellular solution, cell viability was measured using calcein fluorescence.
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