Intracellular ATP (intracellular + atp)

Distribution by Scientific Domains

Terms modified by Intracellular ATP

  • intracellular atp level

  • Selected Abstracts

    LKB1 and AMP-activated protein kinase control of mTOR signalling and growth

    ACTA PHYSIOLOGICA, Issue 1 2009
    R. J. Shaw
    Abstract The AMP-activated serine/threonine protein kinase (AMPK) is a sensor of cellular energy status found in all eukaryotes that is activated under conditions of low intracellular ATP following stresses such as nutrient deprivation or hypoxia. In the past 5 years, work from a large number of laboratories has revealed that one of the major downstream signalling pathways regulated by AMPK is the mammalian target-of-rapamycin [mammalian target of rapamycin (mTOR) pathway]. Interestingly, like AMPK, the mTOR serine/threonine kinase plays key roles not only in growth control and cell proliferation but also in metabolism. Recent work has revealed that across eukaryotes mTOR orthologues are found in two biochemically distinct complexes and only one of those complexes (mTORC1 in mammals) is acutely sensitive to rapamycin and regulated by nutrients and AMPK. Many details of the molecular mechanism by which AMPK inhibits mTORC1 signalling have also been decoded in the past 5 years. AMPK directly phosphorylates at least two proteins to induce rapid suppression of mTORC1 activity, the TSC2 tumour suppressor and the critical mTORC1 binding subunit raptor. Here we explore the molecular connections between AMPK and mTOR signalling pathways and examine the physiological processes in which AMPK regulation of mTOR is critical for growth or metabolic control. The functional conservation of AMPK and TOR in all eukaryotes, and the sequence conservation around the AMPK phosphorylation sites in raptor across all eukaryotes examined suggest that this represents a fundamental cell growth module connecting nutrient status to the cell growth machinery. These findings have broad implications for the control of cell growth by nutrients in a number of cellular and organismal contexts. [source]

    Influence of intracellular Ca2+, mitochondria membrane potential, reactive oxygen species, and intracellular ATP on the mechanism of microcystin-LR induced apoptosis in Carassius auratus lymphocytes in vitro

    H. Zhang
    Abstract Microcystin-LR (MCLR), the most toxic microcystin up to date, could induce apoptosis in many kinds of fish and mammalian cells. For the fish immunotoxicity, it was found that MCLR could induce apoptosis in Carassius auratus lymphocytes in vitro. So this study focused on the role of intracellular Ca2+, mitochondrial membrane potential, reactive oxygen species (ROS), and intracellular ATP in response to the mechanisms of MCLR-induced apoptosis in fish lymphocytes. MCLR (10 nM) administration resulted in a massive elevation in ROS, intracellular Ca2+, decreased ATP, and rapid mitochondrial membrane potential (,,m) disruption. When compared to controls, both a fourfold significant (P < 0.001) elevation in O2, in 1.5 h and an approximately twofold increase in Ca2+ in 0.5 h were observed. After 6 h of treatment, an approximately 30% decrease for ,,m but about 75% decline for ATP were found. Together, the results demonstrated that MCLR-induced apoptosis was associated with a massive calcium influx, resulting in O2, elevation, ,,m disruption, and ATP depletion. This study provided a possible cytotoxic mechanism of fish lymphocytes caused by MCLR. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 559,564, 2007. [source]

    Stabilizing effects of extracellular ATP on synaptic efficacy and plasticity in hippocampal pyramidal neurons

    Eduardo D. Martín
    Abstract The role of adenosine triphosphate (ATP) as a neurotransmitter and extracellular diffusible messenger has recently received considerable attention because of its possible participation in the regulation of synaptic plasticity. However, the possible contribution of extracellular ATP in maintaining and regulating synaptic efficacy during intracellular ATP depletion is understudied. We tested the effects of extracellular ATP on excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by Schaffer collateral stimulation. In the absence of intracellular ATP, EPSC rundown was neutralized when a low concentration of ATP (1 µm) was added to the extracellular solution. Adenosine and ATP analogues did not prevent the EPSC rundown. The P2 antagonists piridoxal-5,-phosphate-azophenyl 2,,4,-disulphonate (PPADS) and reactive blue-2, and the P1 adenosine receptor antagonist 8-cyclopentyltheophylline (CPT) had no detectable effects in cells depleted of ATP. However, the protective action of extracellular ATP on synaptic efficacy was blocked by extracellular application of the protein kinase inhibitors K252b and staurosporine. In contrast, K252b and staurosporine per se did not interfere with synaptic transmission in ATP loaded cells. Without intracellular ATP, bath-applied caffeine induced a transient (< 35 min) EPSC potentiation that was transformed into a persistent long-term potentiation (> 80 min) when 1 µm ATP was added extracellularly. An increased probability of transmitter release paralleled the long-term potentiation induced by caffeine, suggesting that it originated presynaptically. Therefore, we conclude that extracellular ATP may operate to maintain and regulate synaptic efficacy and plasticity in conditions of abnormal intracellular ATP depletion by phosphorylation of a surface protein substrate via activation of ecto-protein kinases. [source]

    A Novel Background Potassium Channel in Rat Atrial Cells

    Z. Shui
    A K+ channel activated by intracellular ATP has been observed in inside-out patches from rat atrial cells. The channel has a slope conductance of 130 ± 5 pS in symmetrical 140 mM K+ solution, and is almost independent of voltage over the range from -80 to +80 mV. There is no detectable inactivation during application of ATP over a few minutes. In the presence of 3 mM intracellular ATP, channel openings occur as bursts with a mean open time of 1.7 ms, a mean closed time of 0.4 ms, a mean burst duration of 18 ms and a mean burst interval of 41 ms. Kinetic analysis suggests that ATP mainly affects the burst duration and the burst interval of the channel. Based on the properties above, the channel differs from other known K+ channels in cardiac cells and may contribute to background K+ current. [source]

    Nitric oxide suppresses transforming growth factor-,1,induced epithelial-to-mesenchymal transition and apoptosis in mouse hepatocytes,

    HEPATOLOGY, Issue 5 2009
    Xinchao Pan
    Nitric oxide (NO) is a multifunctional regulator that is implicated in various physiological and pathological processes. Here we report that administration of NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited transforming growth factor-,1 (TGF-,1)-induced epithelial-to-mesenchymal transition (EMT) and apoptosis in mouse hepatocytes. Overexpression of inducible NO synthase (iNOS) by transfection of the iNOS-expressing vector, which increased NO production, also inhibited the TGF-,1-induced EMT and apoptosis in these cells. Treatment of cells with proinflammatory mediators, including tumor necrosis factor (TNF)-,, interleukin (IL)-1,, and interferon (IFN)-,, which increased the endogenous NO production, produced the same inhibitory effect. Furthermore, exogenous NO donor SNAP treatment caused a decrease in the intracellular adenosine triphosphate (ATP) levels. Consistently, depletion of intracellular ATP by mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited the TGF-,1-induced EMT and apoptosis, suggesting that an NO-induced decrease of ATP involved in the NO-mediated inhibition of TGF-,1-induced EMT and apoptosis. NO and FCCP also inhibited TGF-,1-induced STAT3 activation, suggesting that signal transducer and activator of transcription 3 inactivation is involved in the NO-induced effects on TGF-,1-induced EMT and apoptosis. Conclusion: Our study indicates that NO plays an important role in the inhibition of TGF-,1-induced EMT and apoptosis in mouse hepatocytes through the downregulation of intracellular ATP levels. The data provide an insight into the in vivo mechanisms on the function of NO during the processes of both EMT and apoptosis. (HEPATOLOGY 2009.) [source]

    1-Methyl-4-phenylpridinium (MPP+)-induced functional run-down of GABAA receptor-mediated currents in acutely dissociated dopaminergic neurons

    Jie Wu
    Abstract We have evaluated GABAA receptor function during treatment of 1-methyl-4-phenylpridinium (MPP+) using patch-clamp perforated whole-cell recording techniques in acutely dissociated dopaminergic (DAergic) neurons from rat substantia nigra compacta (SNc). ,-Aminobutyric acid (GABA), glutamate or glycine induced inward currents (IGABA, IGlu, IGly) at a holding potential (VH) of ,45 mV. The IGABA was reversibly blocked by the GABAA receptor antagonist, bicuculline, suggesting that IGABA is mediated through the activation of GABAA receptors. During extracellular perfusion of MPP+ (1,10 ,m), IGABA, but neither IGlu nor IGly, declined (termed run-down) with repetitive agonist applications, indicating that the MPP+ -induced IGABA run-down occurred earlier than IGly or IGlu under our experimental conditions. The MPP+ -induced IGABA run-down can be prevented by a DA transporter inhibitor, mazindol, and can be mimicked by a metabolic inhibitor, rotenone. Using conventional whole-cell recording with different concentrations of ATP in the pipette solution, IGABA run-down can be induced by decreasing intracellular ATP concentrations, or prevented by supplying intracellular ATP, indicating that IGABA run-down is dependent on intracellular ATP concentrations. A GABAA receptor positive modulator, pentobarbital (PB), potentiated the declined IGABA and eliminated IGABA run-down. Corresponding to these patch-clamp data, tyrosine hydroxylase (TH) immunohistochemical staining showed that TH-positive cell loss was protected by PB during MPP+ perfusion. It is concluded that extracellular perfusion of MPP+ induces a functional run-down of GABAA receptors, which may cause an imbalance of excitation and inhibition of DAergic neurons. [source]

    Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells

    Tamás K, szegi
    Abstract Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step. Copyright © 2007 John Wiley & Sons, Ltd. [source]

    Mitochondrial modulation of Ca2+ sparks and transient KCa currents in smooth muscle cells of rat cerebral arteries

    Serguei Y. Cheranov
    Mitochondria sequester and release calcium (Ca2+) and regulate intracellular Ca2+ concentration ([Ca2+]i) in eukaryotic cells. However, the regulation of different Ca2+ signalling modalities by mitochondria in smooth muscle cells is poorly understood. Here, we investigated the regulation of Ca2+ sparks, Ca2+ waves and global [Ca2+]i by mitochondria in cerebral artery smooth muscle cells. CCCP (a protonophore; 1 ,m) and rotenone (an electron transport chain complex I inhibitor; 10 ,m) depolarized mitochondria, reduced Ca2+ spark and wave frequency, and elevated global [Ca2+]i in smooth muscle cells of intact arteries. In voltage-clamped (,40 mV) cells, mitochondrial depolarization elevated global [Ca2+]i, reduced Ca2+ spark amplitude, spatial spread and the effective coupling of sparks to large-conductance Ca2+ -activated potassium (KCa) channels, and decreased transient KCa current frequency and amplitude. Inhibition of Ca2+ sparks and transient KCa currents by mitochondrial depolarization could not be explained by a decrease in intracellular ATP or a reduction in sarcoplasmic reticulum Ca2+ load, and occurred in the presence of diltiazem, a voltage-dependent Ca2+ channel blocker. Ru360 (10 ,m), a mitochondrial Ca2+ uptake blocker, and lonidamine (100 ,m), a permeability transition pore (PTP) opener, inhibited transient KCa currents similarly to mitochondrial depolarization. In contrast, CGP37157 (10 ,m), a mitochondrial Na+,Ca2+ exchange blocker, activated these events. The PTP blockers bongkrekic acid and cyclosporin A both reduced inhibition of transient KCa currents by mitochondrial depolarization. These results indicate that mitochondrial depolarization leads to a voltage-independent elevation in global [Ca2+]i and Ca2+ spark and transient KCa current inhibition. Data also suggest that mitochondrial depolarization inhibits Ca2+ sparks and transient KCa currents via PTP opening and a decrease in intramitochondrial [Ca2+]. [source]

    Nitrite-mediated protection against hypochlorous acid,induced chondrocyte toxicity: A novel cytoprotective role of nitric oxide in the inflamed joint?

    ARTHRITIS & RHEUMATISM, Issue 11 2003
    Matthew Whiteman
    Objective To examine the potential consequences of overproduction of nitric oxide (NO) and nitrite (NO) in the inflamed rheumatoid joint. Methods Human articular chondrocytes in culture were exposed to HOCl (hypochlorous acid, a physiologic oxidant formed in increased amounts at sites of chronic inflammation), and assays of cell viability, intracellular ATP and glutathione (GSH), and lactate dehydrogenase (LDH) were performed. HOCl-induced lipid peroxidation and activation of the MAP kinases ERK-1/2, JNK-1/2, and p38 were also measured. The modulatory effects of NO-derived nitrite (NO) and nitrate (NO) on HOCl-mediated chondrocyte toxicity were investigated. Results Exposure of human articular chondrocytes to HOCl resulted in a concentration- and time-dependent loss of viability, decrease in ATP and GSH levels, LDH leakage, and cell death. HOCl induced significant lipid peroxidation as well as activation of the MAP kinases ERK-1/2 and p38 but not JNK-1/2. However, the presence of NO but not NO substantially decreased HOCl-dependent cellular toxicity even when NO was added at low (,M) concentrations. In sharp contrast, NO (1 mM) did not inhibit superoxide-, hydroxyl radical,, H2O2 -, or peroxynitrite-mediated cytotoxicity. Furthermore, culture media from cells treated with interleukin-1, (to generate NO and NO) offered significantly more protection against HOCl-mediated cytotoxicity than culture media from untreated cells. Conclusion These data suggest that NO accumulation at chronically inflamed sites where both HOCl and NO are overproduced may be cytoprotective against damage induced by HOCl. Accumulation of NO could represent a novel cytoprotective role of NO in inflamed joints. A mechanism for this is suggested. [source]

    NADH supplementation decreases pinacidil-primed IK(ATP) in ventricular cardiomyocytes by increasing intracellular ATP

    Brigitte Pelzmann
    The aim of this study was to investigate the effect of nicotinamide-adenine dinucleotide (NADH) supplementation on the metabolic condition of isolated guinea-pig ventricular cardiomyocytes. The pinacidil-primed ATP-dependent potassium current IK(ATP) was used as an indicator of subsarcolemmal ATP concentration and intracellular adenine nucleotide contents were measured. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36,37°C. Adenine nucleotides were determined by HPLC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPi) IK(ATP) did not contribute to basal electrical activity. The ATP-dependent potassium (K(ATP)) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]i. Supplementation of cardiomyocytes with 300 ,g ml,1 NADH (4,6 h) resulted in a significantly reduced IK(ATP) activation by pinacidil compared to control cells. The current density was 13.8±3.78 (n=6) versus 28.9±3.38 pA pF,1 (n=19; P<0.05). Equimolar amounts of the related compounds nicotinamide and NAD+ did not achieve a similar effect like NADH. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.6±1.88 nmol mg,1 protein versus control: 35.4±2.57 nmol mg,1 protein, P<0.000005). These data show that supplementation of guinea-pig ventricular cardiomyocytes with NADH results in a decreased activation of IK(ATP) by pinacidil compared to control myocytes, indicating a higher subsarcolemmal ATP concentration. Analysis of intracellular adenine nucleotides by HPLC confirmed the significant increase in ATP. British Journal of Pharmacology (2003) 139, 749,754. doi:10.1038/sj.bjp.0705300 [source]

    Functional Roles Of KATP Channels In Vascular Smooth Muscle

    Joseph E Brayden
    SUMMARY 1. ATP-sensitive potassium channels (KATP) are present in vascular smooth muscle cells and play important roles in the vascular responses to a variety of pharmacological and endogenous vasodilators. 2. The KATP channels are composed of four inwardly rectifying K+ channel subunits and four regulatory sulphonylurea receptors. The KATP channels are inhibited by intracellular ATP and by sulphonylurea agents. 3. Pharmacological vasodilators such as cromakalim, pinacidil and diazoxide directly activate KATP channels. The associated membrane hyperpolarization closes voltage-dependent Ca2+ channels, which leads to a reduction in intracellular Ca2+ and vasodilation. 4. Endogenous vasodilators such as calcitonin gene-related peptide, vasoactive intestinal polypeptide, prostacylin and adenosine activate KATP by stimulating the formation of cAMP and increasing the activity of protein kinase A. Part of the mechanism of contraction of endogenous vasoconstrictors is due to inhibition of KATP channels. 5. The KATP channels appear to be tonically active in some vascular beds and contribute to the physiological regulation of vascular tone and blood flow. These channels also are activated under pathophysiological conditions, such as hypoxia, ischaemia, acidosis and septic shock, and, in these disease states, may play an important role in the regulation of tissue perfusion. [source]