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Exogenous ATP (exogenous + atp)
Selected AbstractsPurinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aortaACTA PHYSIOLOGICA, Issue 2 2009S. Hayoz Abstract Aim:, Exogenous ATP elicits a delayed calcium-independent K+ current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. Methods:, The patch-clamp technique was used to record ATP-elicited delayed K+ current in freshly dissociated myocytes. Results:, ATP-elicited delayed K+ current was not inhibited by a ,cocktail' of K+ channel blockers (4-AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K+ current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of ,twin-pore potassium channels' (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K+ current, indicating that P2Y11 is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13-didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real-time quantitative PCR showed that the genes encoding TASK-1 and TASK-2 are expressed. Conclusion:, Our results indicate that blocker cocktail-insensitive delayed K+ current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin-pore channels. [source] Mechanical loading stimulates ecto-ATPase activity in human tendon cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2005M. Tsuzaki Abstract Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5,-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor,ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5,-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5,1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD3 and ENPP1, ENPP2). These data suggest that motion may release ATP from tendon cells in vivo, where ecto-ATPase may also be activated to hydrolyze ATP quickly. Ecto-ATPase may act as a co-modulator in ATP load-signal modulation by regulating the half-life of extracellular purine nucleotides. The extracellular ATP/ATPase system may be important for tendon homeostasis by protecting tendon cells from responding to excessive load signals and activating injurious pathways. © 2005 Wiley-Liss, Inc. [source] Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cellsNEW PHYTOLOGIST, Issue 4 2010Daniela J. Sueldo Summary ,In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca2+, nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. ,In vivo phospholipid signaling upon extracellular ATP treatment was studied in 32Pi -labeled suspension-cultured tomato (Solanum lycopersicum) cells. ,Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mm induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. ,Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase activation. [source] Nitrergic,purinergic interactions in rat distal colon motilityNEUROGASTROENTEROLOGY & MOTILITY, Issue 1 2004K. Van Crombruggen Abstract, Responses of rat distal colon circular muscle strips to exogenous nitric oxide (NO) and adenosine 5,-triphosphate (ATP) and to electrical field stimulation (EFS) were assessed in the absence/presence of various agents that interfere with nitrergic,purinergic pathways. Exogenous NO (10,6 to 10,4 mol L,1) elicited concentration-dependent, tetrodotoxin (TTX)-insensitive relaxations. The soluble guanylyl-cyclase (sGC) inhibitor 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced duration and amplitude; the small conductance Ca2+ -sensitive K+ (SK)-channel blocker apamin (APA) only shortened the relaxations. ODQ + APA showed a marked inhibitory effect on duration and amplitude. TTX, APA, the NO-synthase inhibitor N(omega)-nitro- l -arginine methyl ester (l -NAME) and the purinergic receptor P2Y antagonist Reactive Blue 2 (RB2) shortened the relaxations by exogenous ATP (10,3 mol L,1) but did not influence the amplitude. ODQ had no effect. TTX + l -NAME did not yield a more pronounced inhibitory effect than TTX alone. The effect of ATP- , -S was similar to that of ATP. Electrical field stimulation (EFS) (40 V, 0.05 ms, 0.5,4 Hz for 30 s) yielded TTX-sensitive relaxations that were not altered by l -NAME, ODQ or RB2. APA shortened the relaxations. l -NAME + APA nearly abolished these relaxations. ODQ + APA and RB2 +l -NAME reduced the duration. These results suggest that distinct sets of small conductance SK-channels are involved in the amplitude and the duration of the relaxations and that NO increases their sensitivity to NO and ATP via guanosine 3,,5,-cyclic monophosphate (cGMP). ATP elicits relaxations via P2Y receptors with subsequent activation of SK-channels and induces neuronal release of NO. Both nitrergic and purinergic pathways must be blocked to inhibit EFS-induced relaxations. [source] Extracellular ATP and adenosine induce cell apoptosis of human hepatoma Li-7A cells via the A3 adenosine receptorBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2003Long T Wen Extracellular ATP is a potent signaling molecule that modulates a myriad of cellular functions through the activation of P2 purinergic receptors and is cytotoxic to a variety of cells at higher concentrations. The mechanism of ATP-elicited cytotoxicity is not fully understood. In this study, we investigated the effect of extracellular ATP on the human hepatoma Li-7A cells. We observed a time- and dose-dependent growth inhibition of Li-7A cells by ATP, which is accompanied by an increase in the active form of caspase-3 as well as increased cleavage of its substrate, poly (ADP-ribose) polymerase. The cytotoxic effect of extracellular ATP was not mediated by the P2X7 receptor, since (1) the effect was not abolished by the P2X7 receptor antagonists oxidized ATP and KN-62, and (2) extracellular ADP, AMP, and adenosine were also cytotoxic. We found that ATP and ADP were degraded to adenosine by Li-7A cells and that treatment of Li-7A cells by adenosine resulted in growth inhibition and caspase-3 activation, indicating that adenosine is the apoptotic agent. Using adenosine receptor agonists and antagonists, as well as inhibitors of adenosine transport and deamination, we showed that the cytotoxic effect of adenosine is specifically mediated by the A3 receptor even though transcripts of A1, A2A, A2B, and a splice variant of the P2X7 receptors were detected in Li-7A cells by RT,PCR. Cytotoxicity caused by exogenous ATP and adenosine was completely abolished by the caspase-3 inhibitor Z-DEVD-FMK, demonstrating the central role of caspase-3 in apoptosis of Li-7A cells. British Journal of Pharmacology (2003) 140, 1009,1018. doi:10.1038/sj.bjp.0705523 [source] Expression and function of the purinergic receptor P2X7 in patients with pulmonary tuberculosisCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2006S. Franco-Martínez Summary P2X7 is a channel receptor gated by adenosine triphosphate (ATP) that is involved in the killing of intracellular mycobacteria. To explore further the role of P2X7 in immunity against Mycobacterium tuberculosis, we studied its expression and function in 19 patients with pulmonary tuberculosis (TB) and 19 healthy contacts. Flow cytometry analysis showed a similar and variable expression of P2X7 in TB patients and healthy subjects. In contrast, P2X7 mARN levels were significantly higher in TB patients. When the function of the P2X7 receptor in peripheral blood mononuclear cells (PBMC) was assessed by the effect of exogenous ATP on apoptosis, the uptake of the fluorescent marker Lucifer yellow or extracellular signal regulated kinase (ERK) phosphorylation, no significant differences were detected in patients and controls. However, mRNA macroarray analysis showed that upon stimulation with ATP, the PBMC from TB patients showed a significant induction of a higher number of cytokine genes (27 of 96), and a lower number of apoptosis genes (20 of 96) compared to healthy controls (17 and 76 genes, respectively). These results suggest that although the PBMC from TB patients do not show apparent abnormalities in the expression of P2X7, and the intracellular signals generated through it, the pattern of gene expression induced by ATP in these cells is different from that found in healthy contacts. This phenomenon suggests a defective function of P2X7 in the immune cells from TB patients, a condition that may contribute to the inability of these patients to eliminate the mycobacteria. [source] | |||||||||||||