title: 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) stimulates cellular ATP release through exocytosis of ATP-enriched vesicles.
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.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9151, USA.
authors.forename: Svjetlana
authors.initials: S
authors.lastname: Dolovcak
authors.forename: Shar L
authors.initials: SL
authors.lastname: Waldrop
authors.forename: J Gregory
authors.initials: JG
authors.lastname: Fitz
authors.forename: Gordan
authors.initials: G
authors.lastname: Kilic
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D005089:Exocytosis; D006801:Humans; D007700:Kinetics; D009474:Neurons / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology*; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058486:Receptors, Purinergic P2X7; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D020533:Angiogenesis Inhibitors; D002121:Calcium Channel Blockers; D009579:Nitrobenzoates; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D000255:Adenosine Triphosphate