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.
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ABSTRACT: Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl](L)) and transmit signals to the mesangial cellafferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch-clamp studies of macula densa cells, we identified an [NaCl](L)-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 microM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl](L), by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca(2+) concentration to extracellular application of ATP (EC(50) 0.8 microM). Importantly, we also found increases in cytosolic Ca(2+) concentration with elevations in [NaCl](L), when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y(2) receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.Proceedings of the National Academy of Sciences 05/2003; 100(7):4322-7. · 9.68 Impact Factor
Article: Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors.[show abstract] [hide abstract]
ABSTRACT: The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.Journal of Biological Chemistry 10/2003; 278(39):37344-51. · 4.77 Impact Factor
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ABSTRACT: ATP is released from astrocytes and is involved in the propagation of calcium waves among them. Neuronal ATP secretion is quantal and calcium-dependent, but it has been suggested that ATP release from astrocytes may not be vesicular. Here we report that, besides the described basal ATP release facilitated by exposure to calcium-free medium, astrocytes release purine under conditions of elevated calcium. The evoked release was not affected by the gap-junction blockers anandamide and flufenamic acid, thus excluding purine efflux through connexin hemichannels. Sucrose-gradient analysis revealed that a fraction of ATP is stored in secretory granules, where it is accumulated down an electrochemical proton gradient sensitive to the v-ATPase inhibitor bafilomycin A(1). ATP release was partially sensitive to tetanus neurotoxin, whereas glutamate release from the same intoxicated astrocytes was almost completely impaired. Finally, the activation of metabotropic glutamate receptors, which strongly evokes glutamate release, was only slightly effective in promoting purine secretion. These data indicate that astrocytes concentrate ATP in granules and may release it via a regulated secretion pathway. They also suggest that ATP-storing vesicles may be distinct from glutamate-containing vesicles, thus opening up the possibility that their exocytosis is regulated differently.Journal of Biological Chemistry 02/2003; 278(2):1354-62. · 4.77 Impact Factor