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Sample records for adenosine tri-phosphate atp

  1. Rapid and direct estimation of active biomass on granular activated carbon through adenosine tri-phosphate (ATP) determination.

    PubMed

    Velten, Silvana; Hammes, Frederik; Boller, Markus; Egli, Thomas

    2007-05-01

    Granular activated carbon (GAC) filtration is used during drinking water treatment for the removal of micropollutants such as taste and odour compounds, halogenated hydrocarbons, pesticides and pharmaceuticals. In addition, the active microbial biomass established on GAC is responsible for the removal of biodegradable dissolved organic carbon compounds present in water or formed during oxidation (e.g., ozonation and chlorination) processes. In order to conduct correct kinetic evaluations of DOC removal during drinking water treatment, and to assess the state and performance of full-scale GAC filter installations, an accurate and sensitive method for active biomass determination on GAC is required. We have developed a straight-forward method based on direct measurement of the total adenosine tri-phosphate (ATP) content of a GAC sample and other support media. In this method, we have combined flow-cytometric absolute cell counting and ATP analysis to derive case-specific ATP/cell conversion values. In this study, we present the detailed standardisation of the ATP method. An uncertainty assessment has shown that heterogeneous colonisation of the GAC particles makes the largest contribution to the combined standard uncertainty of the method. The method was applied for the investigation of biofilm formation during the start-up period of a GAC pilot-scale plant treating Lake Zurich water. A rapid increase in the biomass of up to 1.1 x 10(10)cells/g GAC dry weight (DW) within the first 33 days was observed, followed by a slight decrease to an average steady-state concentration of 7.9 x 10(9)cells/g GAC DW. It was shown that the method can be used to determine the biomass attached to the GAC for both stable and developing biofilms.

  2. Unique energetic properties of Adenosine Tri-Phosphate in comparison to similar compounds using density functional theory

    NASA Astrophysics Data System (ADS)

    Muraszko, Kevin; Halloran, Thomas; Malinovskaya, Svetlana; Leopold, Philip

    2015-05-01

    Adenosine Tri-Phosphate (ATP) is arguably the most critical compound to all life known on Earth, serving as the main energy transport and storage in cellular biology. Why in particular did nature ``choose'' ATP instead of a similar compound? We are seeking to answer this question by comparing the energetic properties of ATP to similar compounds. We discuss 3-D models for ATP, variants of the molecule based on all of the separate nucleobases, and ATP's twin molecule Adenosine Di-Phosphate. All calculations were done using Density Functional Theory. The results showed that purine compounds like Adenosine and Guanosine produce similar bond angles, making these viable unlike the other nucleobases. We have analyzed the chiral properties of ATP by comparing the ground-state-energies of ATP-cis and ATP-trans and have shown that ATP-cis is the more energetically favorable of the two. This is consistent with observations in nature.

  3. Flow cytometry and adenosine tri-phosphate analysis: alternative possibilities to evaluate major bacteriological changes in drinking water treatment and distribution systems.

    PubMed

    Vital, Marius; Dignum, Marco; Magic-Knezev, Aleksandra; Ross, Petra; Rietveld, Luuk; Hammes, Frederik

    2012-10-01

    An ever-growing need exists for rapid, quantitative and meaningful methods to quantify and characterize the effect of different treatment steps on the microbiological processes and events that occur during drinking water treatment and distribution. Here we compared cultivation-independent flow cytometry (FCM) and adenosine tri-phosphate (ATP) analysis with conventional cultivation-based microbiological methods, on water samples from two full-scale treatment and distribution systems. The two systems consist of nearly identical treatment trains, but their raw water quality and pre-treatment differed significantly. All of the drinking water treatment processes affected the microbiological content of the water considerably, but once treated, the finished water remained remarkably stable throughout the distribution system. Both the FCM and ATP data were able to describe the microbiology of the systems accurately, providing meaningful process data when combined with other parameters such as dissolved organic carbon analysis. Importantly, the results highlighted a complimentary value of the two independent methods: while similar trends were mostly observed, variations in ATP-per-cell values between water samples were adequately explained by differences in the FCM fingerprints of the samples. This work demonstrates the value of alternative microbial methods for process/system control, optimization and routine monitoring of the general microbial quality of water during treatment and distribution.

  4. Development of a human-specific B. thetaiotaomicron IMS/ATP assay for measuring viable human contamination in surface waters in Baja California, Mexico

    EPA Science Inventory

    Immunomagnetic separation/adenosine triphosphate (IMS/ATP) assays utilize paramagnetic beads and target-specific antibodies to isolate target organisms. Following isolation, adenosine tri-phosphate (ATP) is extracted from the target population and quantified. An inversely-couple...

  5. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  6. Release of Adenosine and ATP During Ischemia and Epilepsy

    PubMed Central

    Dale, Nicholas; Frenguelli, Bruno G

    2009-01-01

    Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions. PMID:20190959

  7. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems.

  8. Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

    PubMed Central

    Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

    2014-01-01

    In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

  9. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

    1975-01-01

    A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

  10. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    PubMed

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

  11. Sawhorse waveform voltammetry for selective detection of adenosine, ATP, and hydrogen peroxide.

    PubMed

    Ross, Ashley E; Venton, B Jill

    2014-08-05

    Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from -0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to -0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo.

  12. Sawhorse Waveform Voltammetry for Selective Detection of Adenosine, ATP, and Hydrogen Peroxide

    PubMed Central

    2015-01-01

    Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from −0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to −0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo. PMID:25005825

  13. K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

    PubMed Central

    Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

    2012-01-01

    BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

  14. Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5'-triphosphate (ATP).

    PubMed

    Sharma, Piyush S; Dabrowski, Marcin; Noworyta, Krzysztof; Huynh, Tan-Phat; Kc, Chandra B; Sobczak, Janusz W; Pieta, Piotr; D'Souza, Francis; Kutner, Wlodzimierz

    2014-09-24

    For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts as such for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5'-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was ∼4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model.

  15. The role of ATP and adenosine in the brain under normoxic and ischemic conditions

    PubMed Central

    Melani, A.; Pugliese, A. M.; Coppi, E.; Cipriani, S.; Traini, C.

    2007-01-01

    By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported. PMID:18404443

  16. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    PubMed

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  17. Enzyme-based field-effect transistor for adenosine triphosphate (ATP) sensing.

    PubMed

    Migita, Satoshi; Ozasa, Kazunari; Tanaka, Tomoya; Haruyama, Tetsuya

    2007-01-01

    Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage.

  18. Endogenous adenosine mediates coronary vasodilation during exercise after K(ATP)+ channel blockade.

    PubMed Central

    Duncker, D J; van Zon, N S; Pavek, T J; Herrlinger, S K; Bache, R J

    1995-01-01

    The mechanism of coronary vasodilation produced by exercise is not understood completely. Recently, we reported that blockade of vascular smooth muscle K(ATP)+ channels decreased coronary blood flow at rest, but did not attenuate the increments in coronary flow produced by exercise. Adenosine is not mandatory for maintaining basal coronary flow, or the increase in flow produced by exercise during normal arterial inflow, but does contribute to coronary vasodilation in hypoperfused myocardium. Therefore, we investigated whether adenosine opposed the hypoperfusion produced by K(ATP)+ channel blockade, thereby contributing to coronary vasodilation during exercise. 11 dogs were studied at rest and during exercise under control conditions, during intracoronary infusion of the K(ATP)+ channel blocker glibenclamide (50 micrograms/kg per min), and during intracoronary glibenclamide in the presence of adenosine receptor blockade. Glibenclamide decreased resting coronary blood flow from 45 +/- 5 to 35 +/- 4 ml/min (P < 0.05), but did not prevent exercise-induced increases of coronary flow. Glibenclamide caused an increase in myocardial oxygen extraction at the highest level of exercise with a decrease in coronary venous oxygen tension from 15.5 +/- 0.7 to 13.6 +/- 0.8 mmHg (P < 0.05). The addition of the adenosine receptor antagonist 8-phenyltheophylline (5 mg/kg intravenous) to K(ATP)+ channel blockade did not further decrease resting coronary blood flow but did attenuate the increase in coronary flow produced by exercise. This was accompanied by a further decrease of coronary venous oxygen tension to 10.1 +/- 0.7 mmHg (P < 0.05), indicating aggravation of the mismatch between oxygen demand and supply. These findings are compatible with the hypothesis that K+ATP channels modulate coronary vasomotor tone both under resting conditions and during exercise. However, when K(ATP)+ channels are blocked, adenosine released from the hypoperfused myocardium provides an alternate

  19. Red blood cells (RBCs), epoxyeicosatrienoic acids (EETs) and adenosine triphosphate (ATP).

    PubMed

    Jiang, Houli; Anderson, Gail D; McGiff, John C

    2010-01-01

    In addition to serving as carriers of O(2), red blood cells (RBCs) regulate vascular resistance and the distribution of microvascular perfusion by liberating adenosine triphosphate (ATP) and epoxyeicosatrienoic acids (EETs) upon exposure to a low O(2) environment. Therefore, RBCs act as sensors that respond to low pO(2) by releasing millimolar amounts of ATP, a signaling molecule, and lipid mediators (EETs). The release of EETs occurs by a mechanism that is activated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, which should greatly amplify the circulatory response to ATP. RBCs are reservoirs of EETs and the primary sources of plasma EETs, which are esterified to the phospholipids of lipoproteins. Levels of free EETs in plasma are low, about 3% of circulating EETs. RBC EETs are produced by direct oxidation of arachidonic acid (AA) esterified to glycerophospholipids and the monooxygenase-like activity of hemoglobin. On release, EETs affect vascular tone, produce profibrinolysis and dampen inflammation. A soluble epoxide hydrolase (sEH) regulates the concentrations of RBC and vascular EETs by metabolizing both cis- and trans-EETs to form dihydroxyeicosatrienoic acids (DHETs). The function and pathophysiological roles of trans-EETs and erythro-DHETs has yet to be integrated into a physiological and pathophysiological context.

  20. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  1. Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats?

    PubMed

    Camara, Henrique; Rodrigues, Juliano Quintella Dantas; Alves, Gabriel Andrade; da Silva Junior, Edilson Dantas; Caricati-Neto, Afonso; Garcia, Antônio G; Jurkiewicz, Aron

    2015-12-05

    Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.

  2. A Comparative Study of Vasorelaxant Effects of ATP, ADP, and Adenosine on the Superior Mesenteric Artery of SHR.

    PubMed

    Watanabe, Shun; Matsumoto, Takayuki; Ando, Makoto; Kobayashi, Shota; Iguchi, Maika; Taguchi, Kumiko; Kobayashi, Tsuneo

    2016-01-01

    We investigated superior mesenteric arteries from spontaneously hypertensive rats (SHR) to determine the relaxation responses induced by ATP, ADP, and adenosine and the relationship between the relaxant effects of these compounds and nitric oxide (NO) or cyclooxygenase (COX)-derived prostanoids. In rat superior mesenteric artery, relaxation induced by ATP and ADP but not by adenosine was completely eliminated by endothelial denudation. In the superior mesenteric arteries isolated from SHR [vs. age-matched control Wistar Kyoto rats (WKY)], a) ATP- and ADP-induced relaxations were weaker, whereas adenosine-induced relaxation was similar in both groups, b) ATP- and ADP-induced relaxations were substantially and partly reduced by N(G)-nitro-L-arginine [a NO synthase (NOS) inhibitor], respectively, c) indomethacin, an inhibitor of COX, increased ATP- and ADP-induced relaxations, d) ADP-induced relaxation was weaker under combined inhibition by NOS and COX, and e) adenosine-induced relaxation was not altered by treatment with these inhibitors. These data indicate that levels of responsiveness to these nucleotides/adenosine vary in the superior mesenteric arteries from SHR and WKY and are differentially modulated by NO and COX-derived prostanoids.

  3. Monitoring of endoscope reprocessing with an adenosine triphosphate (ATP) bioluminescence method

    PubMed Central

    Parohl, Nina; Stiefenhöfer, Doris; Heiligtag, Sabine; Reuter, Henning; Dopadlik, Dana; Mosel, Frank; Gerken, Guido; Dechêne, Alexander; Heintschel von Heinegg, Evelyn; Jochum, Christoph; Buer, Jan; Popp, Walter

    2017-01-01

    Background: The arising challenges over endoscope reprocessing quality proposes to look for possibilities to measure and control the process of endoscope reprocessing. Aim: The goal of this study was to evaluate the feasibility of monitoring endoscope reprocessing with an adenosine triphosphate (ATP) based bioluminescence system. Methods: 60 samples of eight gastroscopes have been assessed from routine clinical use in a major university hospital in Germany. Endoscopes have been assessed with an ATP system and microbial cultures at different timepoints during the reprocessing. Findings: After the bedside flush the mean ATP level in relative light units (RLU) was 19,437 RLU, after the manual cleaning 667 RLU and after the automated endoscope reprocessor (AER) 227 RLU. After the manual cleaning the mean total viable count (TVC) per endoscope was 15.3 CFU/10 ml, and after the AER 5.7 CFU/10 ml. Our results show that there are reprocessing cycles which are not able to clean a patient used endoscope. Conclusion: Our data suggest that monitoring of flexible endoscope with ATP can identify a number of different influence factors, like the endoscope condition and the endoscopic procedure, or especially the quality of the bedside flush and manual cleaning before the AER. More process control is one option to identify and improve influence factors to finally increase the overall reprocessing quality, best of all by different methods. ATP measurement seems to be a valid technique that allows an immediate repeat of the manual cleaning if the ATP results after manual cleaning exceed the established cutoff of 200 RLU.

  4. Effects of adenosine triphosphate (ATP) on early recovery after total knee arthroplasty (TKA): a randomized, double-blind, controlled study.

    PubMed

    Long, Gong; Zhang, Guo Qiang

    2014-12-01

    Functional exercise after total knee arthroplasty (TKA) is necessary. However, it may be a difficult and painful process for the patient. Desirable methods of relieving the patient's pain are worth exploring. Oral supplement of adenosine triphosphate (ATP) is a potential option. In the present study, we decide to investigate whether short-term administration of ATP benefits patients undergoing TKA. A total of 244 subjects were randomized to receive 120mg ATP or placebo each day for 4weeks. Significant differences in quadriceps strength, pain scores at postoperative days 7, 14, 21, and 28 and total opioid consumption were detected. It follows that oral supplement of ATP could benefit patients recovering from TKA.

  5. Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase. adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

    SciTech Connect

    Renosto, F.; Martin, R.L.; Segel, I.H.

    1989-06-05

    At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the (S)0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound (/sup 35/S)APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex.

  6. Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography.

    PubMed

    Zhou, Ling; Xue, XiaoFeng; Zhou, JinHui; Li, Yi; Zhao, Jing; Wu, LiMing

    2012-09-12

    To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.

  7. A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

  8. Raman spectroscopic measurement of base stacking in solutions of adenosine, AMP, ATP, and oligoadenylates.

    PubMed

    Weaver, J L; Williams, R W

    1988-12-13

    Measurements of the colligative properties of nucleosides and their derivatives have shown that bases form transient aggregates in solution [Ts'o (1967) J. Am. Chem. Soc. 89, 3612-3622]. Aggregation of nucleotides cannot be measured by osmometry due to the presence of counterions. Sedimentation measurements are difficult to obtain and have been complicated by differences in pH [Ferguson et al. (1974) Biophys. Chem. 1, 325-337]. Raman studies of oligonucleotides have shown that the intensities due to base vibrational modes depend on the extent of base stacking, but this dependence has not been quantitated. We have measured this dependence by relating changes in the Raman spectra of nucleotides and nucleosides with previous measurements of colligative properties. Visible Raman spectra of ATP, AMP, and adenosine, taken over a range of concentrations from 1 to 1000 mM, show that the peak intensity ratio (I1305 + I1380)/I1340 varies linearly with the log of the concentration for all three bases. This concentration-dependent change correlates with published molal osmotic coefficient data for functionally similar bases with a correlation coefficient of 0.99. In contrast, UV resonance Raman spectra of the same bases show changes that vary linearly with concentration.

  9. The role of microorganisms in the degradation of adenosine triphosphate (ATP) in chill-stored common carp (Cyprinus carpio) fillets.

    PubMed

    Li, Dapeng; Zhang, Longteng; Song, Sijia; Wang, Zhiying; Kong, Chunli; Luo, Yongkang

    2017-06-01

    Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H2S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria.

  10. A sensitive aptasensor for colorimetric detection of adenosine triphosphate based on the protective effect of ATP-aptamer complexes on unmodified gold nanoparticles.

    PubMed

    Huo, Yuan; Qi, Liang; Lv, Xiao-Jun; Lai, Ting; Zhang, Jing; Zhang, Zhi-Qi

    2016-04-15

    Adenosine triphosphate (ATP) is the most direct source of energy in organisms. This study is the first to demonstrate that ATP-aptamer complexes provide greater protection for unmodified gold nanoparticles (AuNPs) against salt-induced aggregation than either aptamer or ATP alone. This protective effect was confirmed using transmission electron microscopy, dynamic light scattering, Zeta potential measurement, and fluorescence polarization techniques. Utilizing controlled particle aggregation/dispersion as a gauge, a sensitive and selective aptasensor for colorimetric detection of ATP was developed using ATP-binding aptamers as the identification element and unmodified AuNPs as the probe. This aptasensor exhibited a good linear relationship between the absorbance and the logarithm concentration of ATP within a 50-1000 nM range. ATP analogs such as guanosine triphosphate, uridine triphosphate and cytidine triphosphate resulted in little or no interference in the determination of ATP.

  11. Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

    PubMed

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

  12. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5⿲ triphosphate (ATP)

    NASA Astrophysics Data System (ADS)

    Hammami, Khaled; El-Feki, Hafed; Marsan, Olivier; Drouet, Christophe

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, ⿦), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m < 1 suggests that the ATP/apatite adsorption process is mostly guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard οGads ° was estimated to only ⿿4 kJ/mol, the large value of Nmax led to significantly negative effective οGads values down to ⿿33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, ⿦).

  13. Sperm morphology, adenosine triphosphate (ATP) concentration and swimming velocity: unexpected relationships in a passerine bird

    PubMed Central

    Bennison, Clair; Brookes, Lola; Slate, Jon; Birkhead, Tim

    2016-01-01

    The relationship between sperm energetics and sperm function is poorly known, but is central to our understanding of the evolution of sperm traits. The aim of this study was to examine how sperm morphology and ATP content affect sperm swimming velocity in the zebra finch Taeniopygia guttata. We exploited the high inter-male variation in this species and created extra experimental power by increasing the number of individuals with very long or short sperm through artificial selection. We found a pronounced quadratic relationship between total sperm length and swimming velocity, with velocity increasing with length up to a point, but declining in the very longest sperm. We also found an unexpected negative association between midpiece length and ATP content: sperm with a short midpiece generally contained the highest concentration of ATP. Low intracellular ATP is therefore unlikely to explain reduced swimming velocity among the very longest sperm (which tend to have a shorter midpiece). PMID:27559067

  14. Ultrasensitive bioluminescent determinations of adenosine triphosphate (ATP) for investigating the energetics of host-grown microbes

    NASA Technical Reports Server (NTRS)

    Hanks, J. H.; Dhople, A. M.

    1975-01-01

    Stability and optimal concentrations of reagents were studied in bioluminescence assay of ATP levels. Luciferase enzyme was prepared and purified using Sephadex G-100. Interdependencies between enzyme and luciferin concentrations in presence of optimal Mg are illustrated. Optimal ionic strength was confirmed to be 0.05 M for the four buffers tested. Adapted features of the R- and H-systems are summarized, as well as the percentages of ATP pools released from representative microbes by heat and chloroform.

  15. Oral adenosine-5’-triphosphate (ATP) administration increases blood flow following exercise in animals and humans

    PubMed Central

    2014-01-01

    Introduction Extracellular adenosine triphosphate (ATP) stimulates vasodilation by binding to endothelial ATP-selective P2Y2 receptors; a phenomenon, which is posited to be accelerated during exercise. Herein, we used a rat model to examine how different dosages of acute oral ATP administration affected the femoral blood flow response prior to, during, and after an exercise bout. In addition, we performed a single dose chronic administration pilot study in resistance trained athletes. Methods Animal study: Male Wistar rats were gavage-fed the body surface area, species adjusted human equivalent dose (HED) of either 100 mg (n=4), 400 mg (n=4), 1,000 mg (n=5) or 1,600 mg (n=5) of oral ATP as a disodium salt (Peak ATP®, TSI, Missoula, MT). Rats that were not gavage-fed were used as controls (CTL, n=5). Blood flow was monitored continuously: a) 60 min prior to, b) during and c) 90 min following an electrically-evoked leg-kicking exercise. Human Study: In a pilot study, 12 college-aged resistance-trained subjects were given 400 mg of ATP (Peak ATP®, TSI, Missoula, MT) daily for 12 weeks, and prior to an acute arm exercise bout at weeks 1, 4, 8, and 12. Ultrasonography-determined volumetric blood flow and vessel dilation in the brachial artery was measured at rest, at rest 30 minutes after supplementation, and then at 0, 3, and 6 minutes after the exercise. Results Animal Study: Rats fed 1,000 mg HED demonstrated significantly greater recovery blood flow (p < 0.01) and total blood flow AUC values (p < 0.05) compared to CTL rats. Specifically, blood flow was elevated in rats fed 1,000 mg HED versus CTL rats at 20 to 90 min post exercise when examining 10-min blood flow intervals (p < 0.05). When examining within-group differences relative to baseline values, rats fed the 1,000 mg and 1,600 mg HED exhibited the most robust increases in blood flow during exercise and into the recovery period. Human study: At weeks 1, 8, and 12, ATP supplementation significantly increased

  16. The effect of growth phase and medium on the use of the firefly adenosine triphosphate (ATP) assay for the quantitation of bacteria

    NASA Technical Reports Server (NTRS)

    Bush, V. N.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was used as a rapid method to determine the number of bacteria in a urine sample after nonbacterial components were removed. Accurate cellular ATP values, determined when bacteria were grown in an environment similar to that in which they were found, were necessary for the calculation of bacterial titer in urine. Cellular ATP values vary depending on the extraction method, the cell growth phase, and cell growth conditions. ATP per cell values of stationary E. coli grown in urine were two times greater than ATP per cell values of cells grown in trypticase soy broth. Glucose and urea were examined as possible components responsible for the cellular ATP variation.

  17. Role of K+ ATP channels and adenosine in the regulation of coronary blood flow during exercise with normal and restricted coronary blood flow.

    PubMed Central

    Duncker, D J; van Zon, N S; Ishibashi, Y; Bache, R J

    1996-01-01

    Regulation of coronary vasomotor tone during exercise is incompletely understood. We investigated the contributions of K+ ATP channels and adenosine to the coronary vasodilation that occurs during exercise in the normal heart and in the presence of a coronary artery stenosis. Dogs that were chronically instrumented with a Doppler flow probe, hydraulic occluder, and indwelling catheter on the left anterior descending coronary artery were exercised on a treadmill to produce heart rates of approximately 200 beats/min. By graded inflation of the occluder to produce a wide range of coronary stenosis severities, we determined the coronary pressure-flow relation. K+ atp channel blockade with intracoronary glibenclamide (10-50 microgram/kg per min) decreased coronary blood flow during exercise at coronary pressures within and below the autoregulatory range, indicating that coronary K+ ATP channel activation is critical for producing coronary vasodilation with either normal arterial inflow or when flow is restricted by a coronary artery stenosis. Adenosine receptor blockade with intravenous 8-phenyltheophylline (5 mg/kg) had no effect on coronary flow at pressures within the autoregulatory range but decreased flow at pressures < 55 mmHg. In contrast, in the presence of K+ ATP channel blockade, the addition of adenosine receptor blockade further decreased coronary flow even at coronary pressures in the autoregulatory range, indicating increased importance of the vasodilator influence of endogenous adenosine during exercise when K+ atp channels are blocked. Intracoronary adenosine (50 microgram/kg per min) increased coronary flow at perfusion pressures both within and below the autoregulatory range. In contrast, selective K+ ATP channel activation with intracoronary pinacidil (0.2-5.0 microgram/kg per min) increased flow at normal but not at lower coronary pressures (< 55 mmHg). This finding demonstrates that not all K+ ATP channels are activated during exercise at pressures

  18. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    DTIC Science & Technology

    1986-01-01

    amphibian sympathetic ganglion to inhibit the M current (8). ATP may affect - . dorsal root terminals in the toad spinal cord (343), and function...Perfusion Twenty-five frogs (Rana pipiens and Rana temporaria) were •de individually sacrificed by decapitation and pithing the spinal cord . During...various nucleosides and nucleotides on the isolated toad spinal cord . Gen. Pharmacol. 9:239-247, 1978. 344. Phillis, J.W. and Wu, P.H. The role of

  19. Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

    PubMed

    Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

    2011-08-17

    The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

  20. Ratiometric detection of adenosine triphosphate (ATP) in water and real-time monitoring of apyrase activity with a tripodal zinc complex.

    PubMed

    Butler, Stephen J

    2014-11-24

    Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.

  1. Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.

    PubMed

    Blundell, Ross D; Williams, Simon J; Arras, Samantha D M; Chitty, Jessica L; Blake, Kirsten L; Ericsson, Daniel J; Tibrewal, Nidhi; Rohr, Jurgen; Koh, Y Q Andre E; Kappler, Ulrike; Robertson, Avril A B; Butler, Mark S; Cooper, Matthew A; Kobe, Bostjan; Fraser, James A

    2016-09-09

    Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

  2. Bcl-2 delays cell cycle through mitochondrial ATP and ROS.

    PubMed

    Du, Xing; Fu, Xufeng; Yao, Kun; Lan, Zhenwei; Xu, Hui; Cui, Qinghua; Yang, Elizabeth

    2017-02-22

    Bcl-2 inhibits cell proliferation by delaying G0/G1 to S phase entry. We tested the hypothesis that Bcl-2 regulates S phase entry through mitochondrial pathways. Existing evidence indicates mitochondrial adenosine tri-phosphate (ATP) and reactive oxygen species (ROS) are important signals in cell survival and cell death, however, the molecular details of how these 2 processes are linked remain unknown. In this study, 2 cell lines stably expressing Bcl-2, 3T3Bcl-2 and C3HBcl-2, and vector-alone PB controls were arrested in G0/G1 phase by serum starvation and contact inhibition, and ATP and ROS were measured during re-stimulation of cell cycle entry. Both ATP and ROS levels were decreased in G0/G1 arrested cells compared with normal growing cells. In addition, ROS levels were significant lower in synchronized Bcl-2 cells than those in PB controls. After re-stimulation, ATP levels increased with time, reaching peak value 1-3 hours ahead of S phase entry for both Bcl-2 cells and PB controls. Consistent with 2 hours of S phase delay, Bcl-2 cells reached ATP peaks 2 hours later than PB control, which suggests a rise in ATP levels is required for S phase entry. To examine the role of ATP and ROS in cell cycle regulation, ATP and ROS level were changed. We observed that elevation of ATP accelerated cell cycle progression in both PB and Bcl-2 cells, and decrease of ATP and ROS to the level equivalent to Bcl-2 cells delayed S phase entry in PB cells. Our results support the hypothesis that Bcl-2 protein regulates mitochondrial metabolism to produce less ATP and ROS, which contributes to S phase entry delay in Bcl-2 cells. These findings reveal a novel mechanistic basis for understanding the link between mitochondrial metabolism and tumor-suppressive function of Bcl-2.

  3. Effect of extracellular adenosine 5'-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability.

    PubMed

    Thuwanut, Paweena; Arya, Nlin; Comizzoli, Pierre; Chatdarong, Kaywalee

    2015-09-15

    Intracellular adenosine 5'-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen-thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris-egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 10(6) sperm vs. 0.002 ± 0.003 μg/2 × 10(6) sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo

  4. Effects of an ATP analogue, adenosine 5'-[α-thio]-triphosphate, on F1-ATPase rotary catalysis, torque generation, and inhibited intermediated formation.

    PubMed

    Yukawa, Ayako; Watanabe, Rikiya; Noji, Hiroyuki

    2015-03-13

    F1-ATPase (F1), an important rotary motor protein, converts the chemical energy of ATP hydrolysis into mechanical energy using rotary motion with extremely high efficiency. The energy-conversion mechanism for this molecular motor has been extensively clarified by previous studies, which indicate that the interactions between the catalytic residues and the β- and γ-phosphates of ATP are indispensable for efficient catalysis and torque generation. However, the role of α-phosphate is largely unknown. In this study, we observed the rotation of F1 fuelled with an ATP analogue, adenosine 5'-[α-thio]-triphosphate (ATPαS), in which the oxygen has been substituted with a sulfur ion to perturb the α-phosphate/F1 interactions. In doing so, we have revealed that ATPαS does not appear to have any impact on the kinetic properties of the motor or on torque generation compared to ATP. On the other hand, F1 was observed to lapse into the ADP-inhibited intermediate states when in the presence of ATPαS more severely than in the presence of ATP, suggesting that the α-phosphate group of ATP contributes to the avoidance of ADP-inhibited intermediate formation.

  5. Evaluation of the Relationship between the Adenosine Triphosphate (ATP) Bioluminescence Assay and the Presence of Bacillus anthracis Spores and Vegetative Cells

    PubMed Central

    Gibbs, Shawn G.; Sayles, Harlan; Colbert, Erica M.; Hewlett, Angela; Chaika, Oleg; Smith, Philip W.

    2014-01-01

    Background: The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. Methods: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. Results: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. Conclusions: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event. PMID:24879485

  6. A1-adenosine acute withdrawal response and cholecystokinin-8 induced contractures are regulated by Ca(2+)- and ATP-activated K(+) channels.

    PubMed

    Cascio, Maria Grazia; Valeri, Daniela; Tucker, Steven J; Marini, Pietro

    2015-01-01

    In isolated guinea-pig ileum (GPI), the A1-adenosine acute withdrawal response is under the control of several neuronal signalling systems, including the μ/κ-opioid and the cannabinoid CB1 systems. It is now well established that after the stimulation of the A1-adenosine system, the indirect activation of both μ/κ-opioid and CB1 systems is prevented by the peptide cholecystokinin-8 (CCk-8). In the present study, we have investigated the involvement of the Ca(2+)/ATP-activated K(+) channels in the regulation of both acute A1-withdrawal and CCk-8-induced contractures in the GPI preparation. Interestingly, we found that: (a) the A1-withdrawal contracture is inhibited by voltage dependent Ca(2+)-activated K(+) channels, Kv, while it is enhanced by the voltage independent Ca(2+)-activated K(+) channels, SKCa; (b) in the presence of CCk-8, the inhibitory effect of the A1 agonist, CPA, on the peptide induced contracture is significantly enhanced by the voltage independent Ca(2+)-activated K(+) channel, SKCa; and (c) the A1-withdrawal contracture precipitated in the presence of CCk-8 is controlled by the ATP-sensitive potassium channels, KATP. Our data suggest, for the first time, that both Ca(2+)- and ATP-activated K(+) channels are involved in the regulation of both A1-withdrawal precipitated and CCk-8 induced contractures.

  7. Development of an immune function assay by measuring intracellular adenosine triphosphate (iATP) levels in mitogen-stimulated CD4+ T lymphocytes.

    PubMed

    Naderi, Hadi; Najafi, Alireza; Khoshroo, Mohammad; Tajik, Nader

    2016-01-01

    We developed an immune function assay for monitoring CD4+ T cells activity based on changes in intracellular adenosine triphosphate (iATP) levels after phytohemagglutinin (PHA) stimulation. Blood samples were obtained from 40 healthy subjects and 30 RTRs and incubated with 5 µg/mL of PHA for 15-18 hr at 37°C and 5% CO2. Afterward, the CD4+ T cells were separated by antibody-coated magnetic beads and lysed. Then, iATP content in unstimulated and stimulated conditions was measured by luciferin-luciferase reaction using a log-log standard curve. The iATP levels showed significant increase in CD4+ T cells in both healthy persons (mean: 550 ± 142 ng/mL vs. 109 ± 54 ng/mL) and RTRs (mean: 394 ± 160 ng/mL vs. 52 ± 37 ng/mL) after PHA stimulation (P < 0.001). However, the iATP production in RTRs was significantly lower than that in healthy individuals; both prior to and after stimulation with PHA (P < 0.001). No gender-specific difference in iATP production was observed between women and men subjects. This rapid and low-cost assay reflects the degree of immune cell function through assessment of CD4+ T cells activation. Thus, it can be used for evaluation of immune system status in immunodeficient individuals as well as in immunosuppressed transplant recipients who needs drug adjustment.

  8. Adenosine Triphosphate (ATP) Inhibits Voltage-Sensitive Potassium Currents in Isolated Hensen's Cells and Nifedipine Protects Against Noise-Induced Hearing Loss in Guinea Pigs.

    PubMed

    Ye, Rui; Liu, Jun; Jia, Zhiying; Wang, Hongyang; Wang, YongAn; Sun, Wei; Wu, Xuan; Zhao, Zhifei; Niu, Baolong; Li, Xingqi; Dai, Guanghai; Li, Jianxiong

    2016-06-13

    BACKGROUND There is increasing evidence that adenosine triphosphate (ATP), a well-known neurotransmitter and neuromodulator in the central nervous system, plays an important role as an extracellular chemical messenger in the cochlea. MATERIAL AND METHODS Using a whole-cell recording technique, we studied the effects of ATP on isolated Hensen's cells, which are supporting cells in the cochlea, to determine if they are involved in the transduction of ions with hair cells. RESULTS ATP (0.1-10 µM) reduced the potassium current (IK+) in the majority of the recorded Hensen's cells (21 out of 25 cells). An inward current was also induced by high concentrations of ATP (100 µM to 10 mM), which was reversibly blocked by 100 µM suramin (a purinergic antagonist) and blocked by nifedipine (an L-type calcium channel blocker). After the cochleas were perfused with artificial perilymph solutions containing nifedipine and exposed to noise, the amplitude increase in the compound action potential (CAP) threshold and the reduction in cochlear microphonics was lower than when they were exposed to noise alone. CONCLUSIONS Our results suggest that ATP can block IK+ channels at a low concentration and induce an inward Ca2+ current at high concentrations, which is reversed by purinergic receptors. Nifedipine may have a partially protective effect on noise-induced hearing loss (NIHL).

  9. An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.

    PubMed

    Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

    2015-01-15

    A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

  10. Extracellular adenosine triphosphate and adenosine in cancer.

    PubMed

    Stagg, J; Smyth, M J

    2010-09-30

    Adenosine triphosphate (ATP) is actively released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P2X and P2Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and dendritic cells, and acts as a co-activator of NLR family, pyrin domain-containing 3 (NLRP3) inflammasomes. The conversion of extracellular ATP to adenosine, in contrast, essentially through the enzymatic activity of the ecto-nucleotidases CD39 and CD73, acts as a negative-feedback mechanism to prevent excessive immune responses. Here we review the effects of extracellular ATP and adenosine on tumorigenesis. First, we summarize the functions of extracellular ATP and adenosine in the context of tumor immunity. Second, we present an overview of the immunosuppressive and pro-angiogenic effects of extracellular adenosine. Third, we present experimental evidence that extracellular ATP and adenosine receptors are expressed by tumor cells and enhance tumor growth. Finally, we discuss recent studies, including our own work, which suggest that therapeutic approaches that promote ATP-mediated activation of inflammasomes, or inhibit the accumulation of tumor-derived extracellular adenosine, may constitute effective new means to induce anticancer activity.

  11. Cilostazol protects the heart against ischaemia reperfusion injury in a rabbit model of myocardial infarction: focus on adenosine, nitric oxide and mitochondrial ATP-sensitive potassium channels.

    PubMed

    Bai, Yushan; Muqier; Murakami, Hiroya; Iwasa, Masamitsu; Sumi, Shohei; Yamada, Yoshihisa; Ushikoshi, Hiroaki; Aoyama, Takuma; Nishigaki, Kazuhiko; Takemura, Genzou; Uno, Bunji; Minatoguchi, Shinya

    2011-10-01

    1. The present study examined whether or not cilostazol reduces the myocardial infarct size, and investigated its mechanism in a rabbit model of myocardial infarction. 2. Japanese white rabbits underwent 30 min of coronary occlusion, followed by 48 h of reperfusion. Cilostazol (1 and 5 mg/kg) or vehicle was given intravenously 5 min before ischaemia. 8-p-sulfophenyl theophylline (8SPT; an adenosine receptor blocker, 7.5 mg/kg), Nω-nitro-L-arginine methylester (l-NAME; an NOS inhibitor, 10 mg/kg) or 5-hydroxydecanoic acid sodium salt (5-HD; a mitochondrial ATP-sensitive potassium (KATP) channel blocker, 5 mg/kg) was given intravenously 5 min before cilostazol injection. Infarct size was determined as a percentage of the risk area. 3. The myocardial interstitial levels of adenosine and nitrogen oxide (NOx) during ischaemia and reperfusion, and the intensity of myocardial dihydroethidium staining were determined. 4. Infarct size was significantly reduced in the cilostazol 1 mg/kg (38.4% (2.9%)) and cilostazol 5 mg/kg (30.7% (4.7%)) groups compared with that in the control group (46.5% (4.2%)). The infarct size-reducing effect of cilostazol was completely abolished by 8SPT (46.6% (3.5%)), L-NAME (49.0% (5.5%)), or 5HD (48.5% (5.1%)). 8SPT, L-NAME or 5HD alone did not affect the infarct size. Cilostazol treatment significantly increased myocardial levels of adenosine and NOx during ischaemia, and attenuated the intensity of dihydroethidium staining during reperfusion. 5. These findings show that cilostazol reduces the myocardial infarct size by increasing adenosine and NOx levels, attenuating superoxide production and opening the mitochondrial KATP channels. Cilostazol might provide a new strategy for the treatment of coronary heart disease.

  12. Rapid and direct detection of attomole adenosine triphosphate (ATP) by MALDI-MS using rutile titania chips.

    PubMed

    Manikandan, Muthu; Hasan, Nazim; Wu, Hui-Fen

    2012-11-07

    We report the rutile titania-based capture of ATP and its application as a MALDI-MS target plate. This chip, when immersed in solutions containing different concentrations of ATP, can capture ATP and lead to its successful detection in MALDI-MS. We have optimized the ideal surface, showing an increased capture efficacy of the 900 °C (rutile) titania surfaces. We demonstrate the use of this chip as a target plate for direct analysis of the attached ATP using MALDI-MS, down to attomolar concentrations. This chip has a promising future for the detection of ATP in environmental samples, which may eventually be used as a pollution indicator in particular environments.

  13. Adenosine triphosphate (ATP) levels in paracetamol-induced cell injury in the rat in vivo and in vitro.

    PubMed

    Martin, F L; McLean, A E

    1995-12-15

    We have investigated the relationship between ATP levels and the onset and progression of cell injury induced by paracetamol overdose both in vivo and in vitro. Liver slices obtained from phenobarbitone-induced and non-induced rats were used in a model in vitro system. Slices were exposed to paracetamol (2-10 mM), for 120 min and then incubated without paracetamol for a further 240 min. ATP levels are reduced upon exposure to paracetamol in liver slices from both phenobarbitone-induced and non-induced rats. Cell injury, as quantified by measuring leakage of lactate dehydrogenase (LDH) and potassium (K+), does not become apparent until 240 min, some 120 min after exposure to paracetamol had ended. This irreversible cell injury is not observed in liver slices from non-induced rats. For in vivo studies rats were phenobarbitone-induced and received i.p. injections of 800 mg/kg body weight paracetamol. Hepatic ATP levels were measured and are found to drop sharply by 3 h post-injection. Development of irreversible hepatic cell injury was assessed by measuring serum enzyme (ALT) activity. ALT levels do not rise until 12 h have elapsed. Paracetamol in overdose gives rise to ATP depletion in liver cells, that is early, independent of paracetamol metabolism and probably spread throughout the lobule. In contrast cell injury is found late and only in our phenobarbitone-induced rats. No cell injury is observed in liver slices from non-induced rats. This suggests that while the level of ATP depletion which is observed may be a necessary part of cell injury by paracetamol, it is not a sufficient cause.

  14. A comparison of certain extracting agents for extraction of adenosine triphosphate (ATP) from microorganisms for use in the firefly luciferase ATP assay

    NASA Technical Reports Server (NTRS)

    Knust, E. A.; Chappelle, E. W.; Picciolo, G. L.

    1975-01-01

    Firefly luciferase ATP assay is used in clinical and industrial applications, such as determination of urinary infection levels, microbial susceptibility testing, and monitoring of yeast levels in beverages. Three categories of extractants were investigated for their extracting efficiency. They were ionizing organic solvents, nonionizing organic solvents, and inorganic acids. Dimethylsulfoxide and formamide represented the ionizing organic solvents, while n-butanol, chloroform, ethanol, acetone, and methylene chloride were used for the nonionizing organic solvents. Nitric acid and perchloric acid were chosen for the inorganic acids category. Pathogens were tested with each solvent. They included: Saccharomyces carlsbergensis, E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, and Candida albicans. These results are shown in graphic representations.

  15. Microbial Group Specific Uptake Kinetics of Inorganic Phosphate and Adenosine-5′-Triphosphate (ATP) in the North Pacific Subtropical Gyre

    PubMed Central

    Björkman, Karin; Duhamel, Solange; Karl, David M.

    2012-01-01

    We investigated the concentration dependent uptake of inorganic phosphate (Pi) and adenosine-5′-triphosphate (ATP) in microbial populations in the North Pacific Subtropical Gyre (NPSG). We used radiotracers to measure substrate uptake into whole water communities, differentiated microbial size classes, and two flow sorted groups; Prochlorococcus (PRO) and non-pigmented bacteria (NPB). The Pi concentrations, uptake rates, and Pi pool turnover times (Tt) were (mean, ±SD); 54.9 ± 35.0 nmol L−1 (n = 22), 4.8 ± 1.9 nmol L−1 day−1 (n = 19), and 14.7 ± 10.2 days (n = 19), respectively. Pi uptake into >2 μm cells was on average 12 ± 7% (n = 15) of the total uptake. The kinetic response to Pi (10–500 nmol L−1) was small, indicating that the microorganisms were close to their maximum uptake velocity (Vmax). Vmax averaged 8.0 ± 3.6 nmol L−1 day−1 (n = 19) in the >0.2 μm group, with half saturation constants (Km) of 40 ± 28 nmol L−1 (n = 19). PRO had three times the cell specific Pi uptake rate of NPB, at ambient concentrations, but when adjusted to cells L−1 the rates were similar, and these two groups were equally competitive for Pi. The Tt of γ-P-ATP in the >0.2 μm group were shorter than for the Pi pool (4.4 ± 1.0 days; n = 6), but this difference diminished in the larger size classes. The kinetic response to ATP was large in the >0.2 μm class with Vmax exceeding the rates at ambient concentrations (mean 62 ± 27 times; n = 6) with a mean Vmax for γ-P-ATP of 2.8 ± 1.0 nmol L−1 day−1, and Km at 11.5 ± 5.4 nmol L−1 (n = 6). The NPB contribution to γ-P-ATP uptake was high (95 ± 3%, n = 4) at ambient concentrations but decreased to ∼50% at the highest ATP amendment. PRO had Km values 5–10 times greater than NPB. The above indicates that PRO and NPB were in close competition in terms of Pi acquisition

  16. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  17. Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides.

    PubMed

    Zhang, Liyuan; Liang, Zhen; Zhang, Lihua; Zhang, Yukui; Shao, Shujuan

    2015-11-05

    Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga(3+) chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga(3+)-ATP-MNPs). The high specificity of Ga(3+)-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga(3+)-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga(3+)-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga(3+)-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga(3+). Moreover, the unique magnetic core of Ga(3+)-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.

  18. Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells

    PubMed Central

    Whittam, R.; Wiley, J. S.

    1968-01-01

    1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts. 2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells. 3. Synthesis of ATP from adenosine plus adenine together was 75% derived from adenine and only 25% from adenosine, indicating that nucleotide synthesis from adenine inhibits the simultaneous synthesis of nucleotide from adenosine. PMID:5723519

  19. Application of the luciferin-luciferase enzyme system for determination of adenosine triphosphate (ATP) to studies on the mechanisms of herbicide action

    NASA Technical Reports Server (NTRS)

    St.john, J. B.

    1975-01-01

    The luciferin-luciferase enzyme system for determination of ATP is valuable for studies on the mechanisms of herbicide action. Investigations using this system have shown that certain herbicides may act by interfering with ATP production or by blocking ATP use, or by both mechanisms.

  20. Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

    SciTech Connect

    Gavel, Olga Yu.; Kladova, Anna V.; Bursakov, Sergey A.; Dias, João M.; Texeira, Susana; Shnyrov, Valery L.; Moura, José J. G.; Moura, Isabel; Romão, Maria J.; Trincão, José

    2008-07-01

    Native zinc-containing ATP sulfurylase from D. desulfuricans ATCC 27774 was purified to homogeneity and crystallized. Diffraction data were collected to 2.5 Å resolution. Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 Å resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

  1. Comparison of the effects of adenine-ribose with adenosine for maintenance of ATP concentrations in 5-day hypothermically perfused dog kidneys.

    PubMed

    McAnulty, J F; Southard, J H; Belzer, F O

    1988-10-01

    The quality of preservation of kidneys is dependent upon a number of factors, one of which may be the concentration of adenine nucleotides in the tissue during long-term perfusion preservation. In this study we have investigated how adenine (5 mM) and ribose (5 mM) in combination affect the concentration of adenine nucleotides in dog kidney cortical tissue after 5 days of continuous hypothermic perfusion preservation. These results were compared to kidneys perfused with adenosine and without any added purine precursors of adenine nucleotide synthesis. Additionally, we investigated how these conditions affected renal tissue slice function after 5 days of preservation and how adenine plus ribose affected renal function after autotransplantation in the dog. Adenosine is nearly completely degraded during 5 days of perfusion but there was little loss of adenine (10%). The adenosine triphosphate concentration in kidney cortical tissue was higher in adenine/ribose-perfused kidneys (1.41 +/- 0.19 mumol/g) than in adenosine-perfused kidneys (0.71 +/- 0.1 mumol/g) after 5 days of preservation. Tissue slices prepared from kidneys preserved in the presence of adenine plus ribose were metabolically more functional (slice volume control and electrolyte pump activity) than slices from adenosine-perfused kidneys. Adenine plus ribose had no detrimental effects on kidneys preserved for 3 days as tested in the autotransplant model but did not yield successful 5-day preservation. Because of some potentially detrimental factors in using adenosine as an adenine nucleotide synthesis precursor, we have now switched to the combination of adenine and ribose for perfusion preservation of kidneys both in the laboratory and in the clinic.

  2. Copper-transporting P-type adenosine triphosphatase (ATP7A) is associated with platinum-resistance in non-small cell lung cancer (NSCLC)

    PubMed Central

    2012-01-01

    Background Copper export protein ATP7A is important for maintaining copper homeostasis. Recent studies have shown that copper transporters are also involved in the transport of platinum. The goal of this study was to determine the role of ATP7A in the platinum-resistance of non-small cell lung cancer (NSCLC). Methods Sensitivities to platinums were detected by MTT assay and drug-resistance related genes were analyzed by real-time PCR and immunoblotting between DDP-sensitive A549 and the corresponding DDP-resistant cell subline (A549/DDP). ATP7A expression was evaluated by immunohistochemistry in tumor tissues of unresectable NSCLC patients who received cisplatin-basing chemotherapy. Results The expression of ATP7A was significantly higher in A549/DDP cell subline than in A549 cells at both mRNA and protein levels. The silencing of ATP7A expression in A549/DDP by siRNA partially reversed DDP-resistance (29.62%) and increased cell apoptosis. ATP7A expression was detected in 41.6%of NSCLC patients, but not in adjacent stroma nor normal lung tissues. ATP7A-positive patients had a significantly poorer histological grade (p = 0.039) and poorer response to platinum-basing chemotherapy (p = 0.001) compared with ATP7A-negative patients. Cox's proportional hazards analysis showed that ATP7A expression was an independent prognostic factor for overall survival (p = 0.045). Conclusions ATP7A overexpression played an important role in platinum-resistance of NSCLC, and was a negative prognostic factor of NSCLC patients treated with platinum-based chemotherapy. PMID:22304828

  3. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  4. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  5. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  6. Potassium Aspartate Attenuates Brain Injury Induced by Controlled Cortical Impact in Rats Through Increasing Adenosine Triphosphate (ATP) Levels, Na+/K+-ATPase Activity and Reducing Brain Edema

    PubMed Central

    Gu, Yi; Zhang, Jie; Zhao, Yumei; Su, Yujin; Zhang, Yazhuo

    2016-01-01

    Background Potassium aspartate (PA), as an electrolyte supplement, is widely used in clinical practice. In our previous study, we found PA had neuroprotective effects against apoptosis after cerebral ischemia/reperfusion in rats. In this study, we examine whether PA has protective effects on traumatic brain injury (TBI). Material/Methods TBI was induced by controlled cortical impact (CCI) in rats. Vehicle treatment (control) or PA treatment was administered intraperitoneally at 30 minutes after CCI. The modified neurological severity score (mNSS) and cortical lesion volume were examined. Brain edema and blood-brain barrier (BBB) integrity were measured, as well as brain ATP contents, lactic acid levels, and Na+/K+-ATPase activities. Results We found that CCI induced cortical injury in rats. Acute PA treatment at the dose of 62.5 mg/kg and 125 mg/kg significantly improved neurological deficits (p<0.05 and p<0.001, respectively) and decreased the cortical lesion volume (p<0.05 and p<0.001, respectively) compared with vehicle-only treatment. PA treatment at the dose of 125 mg/kg attenuated brain edema and ameliorated BBB integrity. In addition, PA treatment significantly reduced the loss of ATP (p<0.01), reduced lactic acid levels (p<0.001), and increased the activity of Na+/K+-ATPase (p<0.01). Conclusions Our results indicate PA has neuroprotective effects on TBI through increasing ATP levels, Na+/K+-ATPase activity, and reducing brain edema. It provides experimental evidence for the clinical application of PA. PMID:27959885

  7. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  8. Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

    PubMed

    Walpole, Thomas B; Palmer, David N; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2015-04-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria.

  9. Conservation of Complete Trimethylation of Lysine-43 in the Rotor Ring of c-Subunits of Metazoan Adenosine Triphosphate (ATP) Synthases*

    PubMed Central

    Walpole, Thomas B.; Palmer, David N.; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2015-01-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9–15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. PMID:25608518

  10. Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity.

    PubMed

    Hung, Szu-Ying; Shih, Ya-Chen; Tseng, Wei-Lung

    2015-02-01

    This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the determination of adenosine in urine.

  11. Continuous intravenous infusion of ATP in humans yields large expansions of erythrocyte ATP pools but extracellular ATP pools are elevated only at the start followed by rapid declines.

    PubMed

    Rapaport, Eliezer; Salikhova, Anna; Abraham, Edward H

    2015-06-01

    The pharmacokinetics of adenosine 5'-triphosphate (ATP) was investigated in a clinical trial that included 15 patients with advanced malignancies (solid tumors). ATP was administered by continuous intravenous infusions of 8 h once weekly for 8 weeks. Three values of blood ATP levels were determined. These were total blood (erythrocyte) and blood plasma (extracellular) ATP pools along with the initial rate of release of ATP into the blood plasma. We found that values related to erythrocyte ATP pools showed great variability (diversity) among individuals (standard deviation of about 30-40% of mean at baseline). It was discovered that erythrocyte baseline ATP pool sizes are unique to each individual and that they fall within a narrow range in each individual. At the end of an 8 h continuous intravenous infusion of ATP, intracellular erythrocyte ATP pools were increased in the range of 40-60% and extracellular ATP declined from elevated levels achieved at the beginning and middle of the infusion, to baseline levels. The ability of erythrocytes to sequester exogenously administered ATP to this degree, after its initial conversion to adenosine in the blood plasma is unexpected, considering that some of the adenosine is likely to have been degraded by in vivo catabolic activities or taken up by organs. The data suggest that administration of ATP by short-term intravenous infusions, of up to 4 h, may be a favorable way for elevating extracellular ATP pools. A large fraction of the total exogenously administered ATP is sequestered into the intracellular compartments of the erythrocytes after an 8 h intravenous infusion. Erythrocytes loaded with ATP are known to release their ATP pools by the application of previously established agents or conditions applied locally or globally to circulating erythrocytes. Rapid degradation of intravenously administered ATP to adenosine and subsequent accumulation of ATP inside erythrocytes indicate the existence of very effective mechanisms

  12. Separation of adenosine diphosphate--adenosine triphosphate-exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase.

    PubMed

    Stahl, W L; Sattin, A; McIlwain, H

    1966-05-01

    1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.

  13. Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

    PubMed Central

    Stahl, W. L.; Sattin, A.; McIlwain, H.

    1966-01-01

    1. A microsomal fraction from ox cerebral cortex catalysed [14C]ADP–ATP exchange at a speed similar to that at which it liberated Pi from ATP in the presence of Na+, K+ and Mg2+. 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na+, K+ or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide–cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na+-plus-K+-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP–ATP-exchange activity does not take part in the Na+-plus-K+-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP–ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na+-plus-K+-stimulated adenosine triphosphatase is indicated. PMID:4223577

  14. Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

    PubMed

    Eguchi, Ryota; Akao, Sanae; Otsuguro, Ken-ichi; Yamaguchi, Soichiro; Ito, Shigeo

    2015-05-01

    Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.

  15. Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection.

    PubMed

    Bushman, Lane R; Kiser, Jennifer J; Rower, Joseph E; Klein, Brandon; Zheng, Jia-Hua; Ray, Michelle L; Anderson, Peter L

    2011-09-10

    An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.

  16. Molecular dynamics simulations and cross-link analysis of the rotary molecular motor F(o) of ATP synthase

    NASA Astrophysics Data System (ADS)

    Kanchanawarin, Chalermpol

    The protein F1Fo ATP synthase is responsible for the generation of the molecule adenosine tri-phosphate (ATP). It consists of two coupled rotary motors, F1 and Fo. ATP is generated via a rotary mechanism that couples the synthesis of ATP in F1 to the proton translocation across Fo which is driven by the electrochemical proton gradient across the membrane. F1 consists of three different subunits in the stoichiometry a1b2c10. So far, only the structure of the c-subunit and a partial structure of the b-subunit have been solved at atomic level detail. However, enough biochemical and structural information is available to construct an atomic model of Fo. There are still many open questions about Fo operation ranging from the subunit arrangement to the proton pathway and proton conduction mechanism to the rotary mechanism that couples the proton conduction to the rotation of its rotor. In this thesis study, I investigated the following four aspects of Fo: the proton pathway in Fo; the motion of F o subunits during its rotation; the rotation of the C-terminal helix of the c-subunit induced by a pH change; the forced rotation of a c10 ring in a membrane. Using molecular dynamics (MD) simulations, I was able to identify a proton pathway formed as two half proton channels in F o, i.e., a proton entrance channel and a proton exit channel. This is in good agreement with experiment. Furthermore, based on the diagramatic cross-link analysis, I propose a new Fo rotary mechanism which shows the cooperative movement of helices in Fo during proton transport and can explain experimental results which could not be explained by previous models. The investigation of the C-terminal helix rotation by NM simulations showed no pH induced rotation of the helix of the c-subunit. Finally, using steered MD simulations, I found that the c10 ring is mechanically robust against forced rotation in vacuum but not in a membrane on the MD time scale. Overall, structural information combined with my

  17. ATP release through pannexon channels

    PubMed Central

    Dahl, Gerhard

    2015-01-01

    Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut. PMID:26009770

  18. Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ 1

    PubMed Central

    Chen, Chong-Maw; Eckert, Richard L.

    1977-01-01

    Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. The phosphorylation of the cytokinin and adenosine required ATP and Mg2+. The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and Vmax for the cytokinin were 31 μm and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 μm and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N6-(δ2-isopentenyl)adenosine. These preparations also phosphorylated N6-(δ2-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present. PMID:16659870

  19. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  1. Metabolic Cooperative Control of Electrolyte Levels by Adenosine Triphosphate in the Frog Muscle

    PubMed Central

    Gulati, J.; Ochsenfeld, M. M.; Ling, G. N.

    1971-01-01

    This study examines the effects of metabolic inhibitors on the content of cellular K, Na, and adenosine triphosphate (ATP). ATP and K are seen to fall in the inhibited tissues. The ATP content is correlated with the K content. The role of ATP is examined according to a recent biophysical approach. It is suggested that ATP may control the electrolyte levels by inducing conformational changes in the cytoplasmic proteins. PMID:5316285

  2. Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, Hordur; Sadler, Martha H.; Hochstein, Lawrence I.

    1986-01-01

    Membranes prepared from various members of the genus Halobacterium contained a Triton X-l00 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90 percent of total protein. The 60-kDa subunit reacted with dicyclohexyl-carbodiimide when inhibition was carried out in an acidic medium. The enzyme from H. saccharovorum, possesses properties of an F(1)F(0) as well as an E(1)E(2) ATPase.

  3. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    PubMed Central

    Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Dias, Ana Sofia; Pelletier, Julie

    2014-01-01

    Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders. PMID:25210228

  4. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

    Ng, Stella; Lim, Hui Si; Ma, Qian; Gao, Zhiqiang

    2016-01-01

    Nucleic acids are among the most researched and applied biomolecules. Their diverse two- and three-dimensional structures in conjunction with their robust chemistry and ease of manipulation provide a rare opportunity for sensor applications. Moreover, their high biocompatibility has seen them being used in the construction of in vivo assays. Various nucleic acid-based devices have been extensively studied as either the principal element in discrete molecule-like sensors or as the main component in the fabrication of sensing devices. The use of aptamers in sensors - aptasensors, in particular, has led to improvements in sensitivity, selectivity, and multiplexing capacity for a wide verity of analytes like proteins, nucleic acids, as well as small biomolecules such as glucose and adenosine triphosphate (ATP). This article reviews the progress in the use of aptamers as the principal component in sensors for optical detection of ATP with an emphasis on sensing mechanism, performance, and applications with some discussion on challenges and perspectives. PMID:27446501

  5. Chemoelectrical energy conversion of adenosine triphosphate

    NASA Astrophysics Data System (ADS)

    Sundaresan, Vishnu Baba; Sarles, Stephen Andrew; Leo, Donald J.

    2007-04-01

    Plant and animal cell membranes transport charged species, neutral molecules and water through ion pumps and channels. The energy required for moving species against established concentration and charge gradients is provided by the biological fuel - adenosine triphosphate (ATP) -synthesized within the cell. The adenosine triphosphatase (ATPases) in a plant cell membrane hydrolyze ATP in the cell cytoplasm to pump protons across the cell membrane. This establishes a proton gradient across the membrane from the cell exterior into the cell cytoplasm. This proton motive force stimulates ion channels that transport nutrients and other species into the cell. This article discusses a device that converts the chemical energy stored in adenosine triphosphate into electrical power using a transporter protein, ATPase. The V-type ATPase proteins used in our prototype are extracted from red beet(Beta vulgaris) tonoplast membranes and reconstituted in a bilayer lipid membrane or BLM formed from POPC and POPS lipids. A pH7 medium that can support ATP hydrolysis is provided on both sides of the membrane and ATP is dissolved in the pH7 buffer on one side of the membrane. Hydrolysis of ATP results in the formation of a phosphate ion and adenosine diphosphate. The energy from the reaction activates ATPase in the BLM and moves a proton across the membrane. The charge gradient established across the BLM due to the reaction and ion transport is converted into electrical current by half-cell reference electrodes. The prototype ATPase cell with an effective BLM area of 4.15 mm2 carrying 15 μl of ATPase proteins was observed to develop a steady state peak power output of 70 nW, which corresponds to a specific power of 1.69 μW/cm2 and a current density of 43.4 μA/cm2 of membrane area.

  6. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for

  7. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  8. A reusable prepositioned ATP reaction chamber

    NASA Technical Reports Server (NTRS)

    Hoffman, D. G.

    1972-01-01

    Luminescence biometer detects presence of life by means of light-emitting chemical reaction of luciferin and luciferase with adenosine triphosphate (ATP) that occurs in all living cells. Amount of light in reaction chamber is measured to determine presence and extent of life.

  9. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  10. The Janus face of adenosine: antiarrhythmic and proarrhythmic actions.

    PubMed

    Szentmiklosi, A József; Galajda, Zoltán; Cseppento, Ágnes; Gesztelyi, Rudolf; Susán, Zsolt; Hegyi, Bence; Nánási, Péter P

    2015-01-01

    Adenosine is a ubiquitous, endogenous purine involved in a variety of physiological and pathophysiological regulatory mechanisms. Adenosine has been proposed as an endogenous antiarrhythmic substance to prevent hypoxia/ischemia-induced arrhythmias. Adenosine (and its precursor, ATP) has been used in the therapy of various cardiac arrhythmias over the past six decades. Its primary indication is treatment of paroxysmal supraventricular tachycardia, but it can be effective in other forms of supraventricular and ventricular arrhythmias, like sinus node reentry based tachycardia, triggered atrial tachycardia, atrioventricular nodal reentry tachycardia, or ventricular tachycardia based on a cAMP-mediated triggered activity. The main advantage is the rapid onset and the short half life (1- 10 sec). Adenosine exerts its antiarrhythmic actions by activation of A1 adenosine receptors located in the sinoatrial and atrioventricular nodes, as well as in activated ventricular myocardium. However, adenosine can also elicit A2A, A2B and A3 adenosine receptor-mediated global side reactions (flushing, dyspnea, chest discomfort), but it may display also proarrhythmic actions mediated by primarily A1 adenosine receptors (e.g. bradyarrhythmia or atrial fibrillation). To avoid the non-specific global adverse reactions, A1 adenosine receptor- selective full agonists (tecadenoson, selodenoson, trabodenoson) have been developed, which agents are currently under clinical trial. During long-term administration with orthosteric agonists, adenosine receptors can be internalized and desensitized. To avoid desensitization, proarrhythmic actions, or global adverse reactions, partial A1 adenosine receptor agonists, like CVT-2759, were developed. In addition, the pharmacologically "silent" site- and event specific adenosinergic drugs, such as adenosine regulating agents and allosteric modulators, might provide attractive opportunity to increase the effectiveness of beneficial actions of adenosine

  11. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  12. Voltage dependence of ATP secretion in mammalian taste cells.

    PubMed

    Romanov, Roman A; Rogachevskaja, Olga A; Khokhlov, Alexander A; Kolesnikov, Stanislav S

    2008-12-01

    Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm-like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca(2+) transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.

  13. Magnetite nanoparticle-induced fluorescence quenching of adenosine triphosphate-BODIPY Conjugates: application to adenosine triphosphate and pyrophosphate sensing.

    PubMed

    Yu, Cheng-Ju; Wu, Su-Mei; Tseng, Wei-Lung

    2013-09-17

    We report that magnetite nanoparticles (Fe3O4 NPs) act as an efficient quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) that is highly fluorescent in bulk solution. BODIPY-ATP molecules attached to the surface of Fe3O4 NPs through the coordination between the triphosphate group of BODIPY-ATP and Fe(3+)/Fe(2+) on the NP surface. The formed complexes induced an apparent reduction in the BODIPY-ATP fluorescence resulting from an oxidative-photoinduced electron transfer (PET) from the BODIPY-ATP excited state to an unfilled d shell of Fe(3+)/Fe(2+) on the NP surface. A comparison of the Stern-Volmer quenching constant between Fe(3+) and Fe(2+) suggests that Fe(3+) on the NP surface dominantly controls this quenching process. The efficiency for Fe3O4 NP-induced fluorescence quenching of the BODIPY-ATP was enhanced by increasing the concentration of Fe3O4 NPs and lowering the pH of the solution to below 6.0. We found that pyrophosphate and ATP compete with BODIPY-ATP for binding to Fe3O4 NPs. Thus, we amplified BODIPY-ATP fluorescence in the presence of increasing the pyrophosphate and ATP concentration; the detection limits at a signal-to-noise ratio of 3 for pyrophosphate and ATP were determined to be 7 and 30 nM, respectively. The Fe3O4 NP-based competitive binding assay detected ATP and pyrophosphate in only 5 min. The selectivity of this assay for ATP over metal ions, amino acids, and adenosine analogues is particularly high. The practicality of using the developed method to determine ATP in a single drop of blood is also validated.

  14. Mechanical stimulation evokes rapid increases in extracellular adenosine concentration in the prefrontal cortex.

    PubMed

    Ross, Ashley E; Nguyen, Michael D; Privman, Eve; Venton, B Jill

    2014-07-01

    Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon-fiber microelectrodes and fast-scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15-μm-diameter glass pipette near the carbon-fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50-μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM-1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective. We have discovered immediate changes in adenosine concentration in the prefrontal cortex following mechanical stimulation. The adenosine increase lasts only about 20 s. Mechanically stimulated adenosine was activity dependent and mostly because of extracellular ATP metabolism. This rapid, transient increase in adenosine may help protect tissue and would occur during implantation of any electrode, such as during deep brain stimulation.

  15. Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

    PubMed

    Shah, Mrugeshkumar K; Kadowitz, Philip J

    2002-01-01

    The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.

  16. Unpredictable Chronic Stress Alters Adenosine Metabolism in Zebrafish Brain.

    PubMed

    Zimmermann, F F; Altenhofen, S; Kist, L W; Leite, C E; Bogo, M R; Cognato, G P; Bonan, C D

    2016-05-01

    Stress is considered a risk factor for several human disorders. Despite the broad knowledge of stress responses in mammals, data on the relationship between unpredictable chronic stress (UCS) and its effects on purinergic signaling are limited. ATP hydrolysis by ectonucleotidases is an important source of adenosine, and adenosine deaminase (ADA) contributes to the control of the nucleoside concentrations. Considering that some stress models could affect signaling systems, the objective of this study was to investigate whether UCS alters ectonucleotidase and ADA pathway in zebrafish brain. Additionally, we analyzed ATP metabolism as well as ada1, ada2.1, ada2.2, adaL, and adaasi gene expression in zebrafish brain. Our results have demonstrated that UCS did not alter ectonucleotidase and soluble ADA activities. However, ecto-ADA activity was significantly decreased (26.8%) in brain membranes of animals exposed to UCS when compared to the control group. Quantitative reverse transcription PCR (RT-PCR) analysis did not show significant changes on ADA gene expression after the UCS exposure. The brain ATP metabolism showed a marked increase in adenosine levels (ADO) in animals exposed to UCS. These data suggest an increase on extracellular adenosine levels in zebrafish brain. Since this nucleoside has neuromodulatory and anxiolytic effects, changes in adenosine levels could play a role in counteracting the stress, which could be related to a compensatory mechanism in order to restore the homeostasis.

  17. ATP-triggered anticancer drug delivery

    NASA Astrophysics Data System (ADS)

    Mo, Ran; Jiang, Tianyue; Disanto, Rocco; Tai, Wanyi; Gu, Zhen

    2014-03-01

    Stimuli-triggered drug delivery systems have been increasingly used to promote physiological specificity and on-demand therapeutic efficacy of anticancer drugs. Here we utilize adenosine-5'-triphosphate (ATP) as a trigger for the controlled release of anticancer drugs. We demonstrate that polymeric nanocarriers functionalized with an ATP-binding aptamer-incorporated DNA motif can selectively release the intercalating doxorubicin via a conformational switch when in an ATP-rich environment. The half-maximal inhibitory concentration of ATP-responsive nanovehicles is 0.24 μM in MDA-MB-231 cells, a 3.6-fold increase in the cytotoxicity compared with that of non-ATP-responsive nanovehicles. Equipped with an outer shell crosslinked by hyaluronic acid, a specific tumour-targeting ligand, the ATP-responsive nanocarriers present an improvement in the chemotherapeutic inhibition of tumour growth using xenograft MDA-MB-231 tumour-bearing mice. This ATP-triggered drug release system provides a more sophisticated drug delivery system, which can differentiate ATP levels to facilitate the selective release of drugs.

  18. Intracellular Assessment of ATP Levels in Caenorhabditis elegans

    PubMed Central

    Palikaras, Konstantinos; Tavernarakis, Nektarios

    2017-01-01

    Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans. PMID:28194429

  19. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  20. Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes.

    PubMed Central

    Bontemps, F; Van den Berghe, G; Hers, H G

    1983-01-01

    The effect of adenosine on the metabolism of prelabeled adenine nucleotides was investigated in isolated hepatocytes. Adenosine caused an approximately equal to 2-fold increase in the ATP content of the cells. This effect was in part counteracted by an increased rate of adenine nucleotide catabolism that could be explained by a stimulation of both AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) and the cytoplasmic 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) because of the increased concentration of ATP. The unexpected finding that labeled adenosine was formed immediately after the addition of the unlabeled nucleoside could be explained by the trapping effect of adenosine. An accumulation of labeled adenosine was observed also in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). Under these conditions, there was a decrease in the concentration of ATP in the cell and a 2- to 3-fold increase in the rate of formation of allantoin. This formation of adenosine was only slightly decreased by inhibition of the membranous 5'-nucleotidase; it led to the accumulation of S-adenosylhomocysteine in the presence of coformycin and an excess of L-homocysteine. It was concluded that, under basal conditions, the cytoplasmic 5'-nucleotidase present in the liver cell continuously produces adenosine, which is immediately reconverted into AMP by adenosine kinase, without giving rise to allantoin. This futile cycle between AMP and adenosine amounts to at least 20 nmol/min per g of liver and, thus, exceeds the basic rate of allantoin formation. PMID:6304684

  1. Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells.

    PubMed Central

    Ogilvie, A; Lüthje, J; Pohl, U; Busse, R

    1989-01-01

    The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A. PMID:2541689

  2. Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

    PubMed Central

    Sayós, J; Solsona, C; Mallol, J; Lluis, C; Franco, R

    1994-01-01

    Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney. PMID:8110185

  3. Role of adenosine signalling and metabolism in β-cell regeneration

    SciTech Connect

    Andersson, Olov

    2014-02-01

    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.

  4. Determination of adenosine triphosphate on marine particulates: synthesis of methods for use on OTEC samples

    SciTech Connect

    Jones, A.T.; Hartwig, E.O.

    1982-08-01

    Adenosine triphosphate (ATP) is an indicator of living biomass in marine particulates. This report details the method used by Lawrence Berkeley Laboratory to analyze particulate ATP in samples taken from oligotrophic, tropical ocean waters. It represents a synthesis of previously published methods.

  5. Determination of Adenosine Triphosphate on Marine Particulates:Synthesis of Methods for Use on OTEC Samples

    SciTech Connect

    Jones, Anthony T.; Hartwig, Eric O.

    1982-08-01

    Adenosine triphosphate (ATP) is an indicator of living biomass in marine particulates. This report details the method used by Lawrence Berkeley Laboratory to analyze particulate ATP in samples taken from oligotrophic, tropical ocean waters. It represents a synthesis of previously published methods.

  6. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  7. Temporal variations of adenosine metabolism in human blood.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Aguilar-Roblero, R; Oksenberg, A; Vega-González, A; Villalobos, L; Rosenthal, L; Fernández-Cancino, F; Drucker-Colín, R; Díaz-Muñoz, M

    1996-08-01

    Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism.

  8. The breakdown of adenosine triphosphate in the contraction cycle of the frog sartorius muscle

    PubMed Central

    Mommaerts, W. F. H. M.; Wallner, A.

    1967-01-01

    1. It is confirmed that a fluorodinitrobenzene (FDNB)-treated frog sartorius muscle does not split phosphorylcreatine in the course of its contraction cycle, but does use adenosine triphosphate (ATP). 2. Good stoicheiometric relations between the diminution of ATP and the formation of adenosine diphosphate (ADP), adenosine monophosphate (AMP) and phosphate are obtained, and in a 0·2 sec tetanus at 0° C the net break-down of ATP amounts to 0·27, the total equivalent break-down to 0·34 μmoles/g. 3. There is no difference in this quantity between muscles interrupted at the height of contraction and those that have also relaxed, and, in experiments specifically designed to determine relaxation metabolism separately, no such metabolism is found. Thus, all the ATP-break-down occurs in the contraction phase. PMID:6065882

  9. Microcontroller-assisted compensation of adenosine triphosphate levels: instrument and method development.

    PubMed

    Hu, Jie-Bi; Chen, Ting-Ru; Chen, Yu-Chie; Urban, Pawel L

    2015-01-30

    In order to ascertain optimum conditions for biocatalytic processes carried out in vitro, we have designed a bio-opto-electronic system which ensures real-time compensation for depletion of adenosine triphosphate (ATP) in reactions involving transfer of phosphate groups. The system covers ATP concentration range of 2-48 μM. The report demonstrates feasibility of the device operation using apyrase as the ATP-depleting enzyme.

  10. Extraction and quantification of adenosine triphosphate in mammalian tissues and cells.

    PubMed

    Chida, Junji; Kido, Hiroshi

    2014-01-01

    Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.

  11. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release

    PubMed Central

    Nguyen, Michael D.; Venton, B. Jill

    2014-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future. PMID:26900429

  12. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release.

    PubMed

    Nguyen, Michael D; Venton, B Jill

    2015-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future.

  13. Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta

    SciTech Connect

    Kroll, K.; Kelm, M.K.; Buerrig, K.F.S.; Schrader, J.

    1989-06-01

    This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1%/hr of total endothelial radioactivity.

  14. Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5' -phosphoramidate from adenosine 5' -phosphosulphate and ammonia.

    PubMed

    Fankhauser, H; Schiff, J A; Garber, L J

    1981-06-01

    Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5'-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5'-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5'-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5'-phosphoramidate. The enzyme that catalyses the formation of adenosine 5'-phosphoramidate from ammonia and adenosine 5'-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH(4))(2)SO(4) precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2-agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000-65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3'-phosphate 5'-phosphosulphate will not replace adenosine 5'-phosphosulphate, and the apparent K(m) for the last-mentioned compound is 0.82mm. The apparent K(m) for ammonia (assuming NH(3) to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5'-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5'-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme

  15. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  16. Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine.

    PubMed

    Naguib, Fardos N M; Rais, Reem H; Al Safarjalani, Omar N; el Kouni, Mahmoud H

    2015-10-01

    Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo=0.002±0.0002 mM, KATP=0.05±0.008 mM, and Vmax=920±35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.

  17. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    Oldham, Michael L.; Chen, Jue

    2011-12-05

    ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

  18. Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

    PubMed Central

    Sims, Robert Edward; Dale, Nicholas

    2014-01-01

    In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep. PMID:24489921

  19. Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

    PubMed

    Lik-Shing Tsang, M; Schiff, J A

    1976-05-17

    Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.

  20. Detection of ATP and NADH: A Bioluminescent Experience.

    ERIC Educational Resources Information Center

    Selig, Ted C.; And Others

    1984-01-01

    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  1. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    ERIC Educational Resources Information Center

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  2. Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine-5'-phosphosulfate (APS) from sulfate and ATP. To better unde...

  3. Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs.

    PubMed

    Hacker, Stephan M; Buntz, Annette; Zumbusch, Andreas; Marx, Andreas

    2015-11-20

    Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells.

  4. Detecting adenosine triphosphate in the pericellular space.

    PubMed

    Falzoni, Simonetta; Donvito, Giovanna; Di Virgilio, Francesco

    2013-06-06

    Release of adenosine triphosphate (ATP) into the extracellular space occurs in response to a multiplicity of physiological and pathological stimuli in virtually all cells and tissues. A role for extracellular ATP has been identified in processes as different as neurotransmission, endocrine and exocrine secretion, smooth muscle contraction, bone metabolism, cell proliferation, immunity and inflammation. However, ATP measurement in the extracellular space has proved a daunting task until recently. To tackle this challenge, some years ago, we designed and engineered a novel luciferase probe targeted to and expressed on the outer aspect of the plasma membrane. This novel probe was constructed by appending to firefly luciferase the N-terminal leader sequence and the C-terminal glycophosphatidylinositol anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase, is targeted and localized to the outer side of the plasma membrane. With this probe, we have generated stably transfected HEK293 cell clones that act as an in vitro and in vivo sensor of the extracellular ATP concentration in several disease conditions, such as experimentally induced tumours and inflammation.

  5. Hyperpolization-activated Ca(2+) channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba.

    PubMed

    Wang, Fang; Jia, Juanjuan; Wang, Yufang; Wang, Weixia; Chen, Yuling; Liu, Ting; Shang, Zhonglin

    2014-09-01

    Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5'-O-(3-thio) triphosphate (ATPγS), 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5'-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca(2+) channel blockers GdCl3 and LaCl3. A hyperpolarization-activated Ca(2+) channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca(2+) channel significantly. Extracellular ATP-promoted Ca(2+) channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca(2+) channels.

  6. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  7. ATP and related purines stimulate motility, spatial congregation, and coalescence in red algal spores.

    PubMed

    Huidobro-Toro, Juan P; Donoso, Verónica; Flores, Verónica; Santelices, Bernabé

    2015-04-01

    Adenosine 5'-triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration-response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time-dependent manner. 10-300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2-2.5 μm · s(-1) . 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7-fold as compared with parallel controls. Likewise, 1-10 μM cAMP or 30-100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.

  8. ATP-sulfurylase, sulfur-compounds, and plant stress tolerance.

    PubMed

    Anjum, Naser A; Gill, Ritu; Kaushik, Manjeri; Hasanuzzaman, Mirza; Pereira, Eduarda; Ahmad, Iqbal; Tuteja, Narendra; Gill, Sarvajeet S

    2015-01-01

    Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO4 (2-)), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO4 (2-)-activation and yields activated high-energy compound adenosine-5(')-phosphosulfate that is reduced to sulfide (S(2-)) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes.

  9. Release of extracellular ATP by bacteria during growth

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate (ATP) is used as an intracellular energy source by all living organisms. It plays a central role in the respiration and metabolism, and is the most important energy supplier in many enzymatic reactions. Its critical role as the energy storage molecule makes it extremely valuable to all cells. Results We report here the detection of extracellular ATP in the cultures of a variety of bacterial species. The levels of the extracellular ATP in bacterial cultures peaked around the end of the log phase and decreased in the stationary phase of growth. Extracellular ATP levels were dependent on the cellular respiration as bacterial mutants lacking cytochrome bo oxidase displayed lower extracellular ATP levels. We have also shown that Escherichia coli (E. coli) and Salmonella actively depleted extracellular ATP and an ATP supplement in culture media enhanced the stationary survival of E. coli and Salmonella. In addition to E. coli and Salmonella the presence of the extracellular ATP was observed in a variety of bacterial species that contain human pathogens such as Acinetobacter, Pseudomonas, Klebsiella and Staphylococcus. Conclusion Our results indicate that extracellular ATP is produced by many bacterial species during growth and extracellular ATP may serve a role in the bacterial physiology. PMID:24364860

  10. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

  11. Radioprotective effects of ATP in human blood ex vivo

    SciTech Connect

    Swennen, Els L.R. Dagnelie, Pieter C.; Van den Beucken, Twan; Bast, Aalt

    2008-03-07

    Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-{alpha} release and increased IL-10 release. The inhibitory effect of ATP on TNF- {alpha} release was completely reversed by adenosine 5'-O-thiomonophosphate, indicating a P2Y{sub 11} mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6 h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.

  12. Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy.

    PubMed

    Doná, Flávia; Conceição, Isaltino Marcelo; Ulrich, Henning; Ribeiro, Eliane Beraldi; Freitas, Thalma Ariani; Nencioni, Ana Leonor Abrahao; da Silva Fernandes, Maria José

    2016-06-01

    Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.

  13. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  14. Sustained release carrier for adenosine triphosphate as signaling molecule.

    PubMed

    Wischke, Christian; Weigel, Judith; Bulavina, Larisa; Lendlein, Andreas

    2014-12-10

    Adenosine triphosphate (ATP) is a molecule with a fascinating variety of intracellular and extracellular biological functions that go far beyond energy metabolism. Due to its limited passive diffusion through biological membranes, controlled release systems may allow to interact with ATP-mediated extracellular processes. In this study, two release systems were explored to evaluate the capacity for either long-term or short-term release: (i) Poly[(rac-lactide)-co-glycolide] (PLGA) implant rods were capable of ATP release over days to weeks, depending on the PLGA molecular weight and end-group capping, but were also associated with partial hydrolytic degradation of ATP to ADP and AMP, but not adenosine. (ii) Thermosensitive methylcellulose hydrogels with a gelation occurring at body temperature allowed combining adjustable loading levels and the capacity for injection, with injection forces less than 50N even for small 27G needles. Finally, a first in vitro study illustrated purinergic-triggered response of primary murine microglia to ATP released from hydrogels, demonstrating the potential relevance for biomedical applications.

  15. Effects of adenosine metabolism in astrocytes on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-liang; Zhang, Ya-nan; Wang, Zhong-zhuang; Xu, Wei-gang; Li, Run-ping; Zhang, Jun-dong

    2016-03-15

    Hyperbaric oxygen (HBO) is widely used in military operations, especially underwater missions. However, prolonged and continuous inhalation of HBO can cause central nervous system oxygen toxicity (CNS-OT), which greatly limits HBO's application. The regulation of astrocytes to the metabolism of adenosine is involved in epilepsy. In our study, we aimed to observe the effects of HBO exposure on the metabolism of adenosine in the brain. Furthermore, we aimed to confirm the possible mechanism underlying adenosine's mediation of the CNS-OT. Firstly, anesthetized rats exposed to 5 atm absolute HBO for 80 min. The concentrations of extracellular adenosine, ATP, ADP, and AMP were detected. Secondly, free-moving rats were exposed to HBO at the same pressure for 20 min, and the activities of 5'-nucleotidase and ADK in brain tissues were measured. For the mechanism studies, we observed the effects of a series of different doses of drugs related to adenosine metabolism on the latency of CNS-OT. Results showed HBO exposure could increase adenosine content by inhibiting ADK activity and improving 5'-nucleotidase activity. And adenosine metabolism during HBO exposure may be a protective response against HBO-induced CNS-OT. Moreover, the improvement of adenosine concentration, activation of adenosine A1R, or suppression of ADK and adenosine A2AR, which are involved in the prevention of HBO-induced CNS-OT. This is the first study to demonstrate HBO exposure regulated adenosine metabolism in the brain. Adenosine metabolism and adenosine receptors are related to HBO-induced CNS-OT development. These results will provide new potential targets for the termination or the attenuation of CNS-OT.

  16. Why do premature newborn infants display elevated blood adenosine levels?

    PubMed

    Panfoli, Isabella; Cassanello, Michela; Bruschettini, Matteo; Colella, Marina; Cerone, Roberto; Ravera, Silvia; Calzia, Daniela; Candiano, Giovanni; Ramenghi, Luca

    2016-05-01

    Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW

  17. Effects of adenosine triphosphate concentration on motor force regulation during skeletal muscle contraction

    NASA Astrophysics Data System (ADS)

    Wei, J.; Dong, C.; Chen, B.

    2017-03-01

    We employ a mechanical model of sarcomere to quantitatively investigate how adenosine triphosphate (ATP) concentration affects motor force regulation during skeletal muscle contraction. Our simulation indicates that there can be negative cross-bridges resisting contraction within the sarcomere and higher ATP concentration would decrease the resistance force from negative cross-bridges by promoting their timely detachment. It is revealed that the motor force is well regulated only when ATP concentration is above a certain level. These predictions may provide insights into the role of ATP in regulating coordination among multiple motors.

  18. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  19. Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate12

    PubMed Central

    Feng, Lili; Sun, Xiaofeng; Csizmadia, Eva; Han, Lihui; Bian, Shu; Murakami, Takashi; Wang, Xin; Robson, Simon C; Wu, Yan

    2011-01-01

    Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5′-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X7 receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X7 expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management. PMID:21390184

  20. Adenosine induces apoptosis through TNFR1/RIPK1/P38 axis in colon cancer cells.

    PubMed

    Yu, Shunji; Hou, Daisen; Chen, Ping; Zhang, Qi; Lv, Bin; Ma, Yunfang; Liu, Fuchen; Liu, Hui; Song, Evelyn J; Yang, Dongqin; Liu, Jie

    2015-05-08

    Adenosine, a metabolite of ATP, ubiquitously exists in a wide range of organs and tissues. We previously reported that adenosine was implicated in apoptosis in many cancer cells by extrinsic and/or intrinsic pathways. Here, we found that adenosine suppresses the cell growth by induction of apoptosis of human colonic cancer cells through a novel mechanism. Adenosine suppresses the cell growth of human SW620 and SW480 colon cells in an adenosine transporter and adenosine kinase dependent manner. Moreover, the cell growth suppression is induced by apoptosis through activation of caspase-3 and PARP, and accumulation of ROS in cells. Importantly, we found that adenosine increases the expression of TNFR1 and RIPK1 and the phosphorylation of p38. Knockdown of TNFR1 or RIPK1 impairs the activation of p38, blocks the cleavage of PARP, and provides partially, yet significantly protection from cell death, including reducing the ROS generation in the colon cancer cells. These results indicate that a TNFR1/RIPK1/P38 axis is present in adenosine-induced apoptosis of colonic cancer cells. This axis triggers apoptosis and plays crucial roles in relay of the death signaling. Our study also provides additional experimental evidence for adenosine as a potent therapeutic drug in cancer therapy.

  1. Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

    PubMed Central

    Daddona, P E; Mitchell, B S; Meuwissen, H J; Davidson, B L; Wilson, J M; Koller, C A

    1983-01-01

    In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:6603477

  2. Modulation by extracellular ATP of L-type calcium channels in guinea-pig single sinoatrial nodal cell.

    PubMed Central

    Qi, A. D.; Kwan, Y. W.

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate ([ATP]zero) on the L-type Ca2+ channel currents in guinea-pig single sinoatrial nodal (SAN) cells, isolated by enzymatic dissociation, were investigated by use of whole-cell patch-clamp techniques. 2. The application of [ATP]zero (2 microM-1 mM) produced an inhibitory effect on the L-type Ca2+ channel current peak amplitude (10 mM Ba2+ as charge carrier) in a concentration-dependent and reversible manner with an IC50 of 100 microM and a Hill coefficient of 1.83. 3. The presence of the adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM) and 8-phenyltheophylline (10 microM) did not affect the [ATP]zero-induced inhibition of the Ca2+ channel currents. Adenosine (100 microM) had little effect on the basal Ca2+ channel currents. Adenosine 500 microM, caused 23% inhibition of the Ca2+ channel current, which was abolished by 0.1 microM DPCPX. 4. The presence of the P2-purinoceptor antagonists, suramin (1, 10 and 100 microM), reactive blue 2 (1 and 10 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 50 and 100 microM) failed to affect the inhibitory action of [ATP]zero on Ca2+ channel currents. 5. The relative rank order of potency of different nucleotides and nucleosides, at a concentration of 100 microM, on the inhibition of the Ca2+ channel currents is as follows: adenosine 5'-triphosphate (ATP) = alpha,beta-methylene-ATP (alpha,beta MeATP) > > 2-methylthioATP (2-MeSATP) > or = adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > > uridine 5'-triphosphate (UTP) = adenosine 5'-diphosphate (ADP) > adenosine 5'-monophosphate (AMP) > or = adenosine. 6. These results suggest that [ATP]zero may play an important role in the heart beat by inhibiting the L-type Ca2+ channel currents in single SAN cells. This inhibitory effect is not due to the formation of adenosine resulting from the enzymatic degradation of [ATP]zero. Based on the relative order of inhibitory

  3. Imaging changes in the cytosolic ATP-to-ADP ratio

    PubMed Central

    Tantama, Mathew; Yellen, Gary

    2015-01-01

    Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We therefore developed a genetically-encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy. PMID:25416365

  4. Ameliorative effect of adenosine on hypoxia-reoxygenation injury in LLC-PK1, a porcine kidney cell line.

    PubMed

    Yonehana, T; Gemba, M

    1999-06-01

    We studied the effects of adenosine on injury caused by hypoxia and reoxygenation in LLC-PK1 cells. Lactate dehydrogenase and gamma-glutamyltranspeptidase were released from cells exposed to hypoxia for 6 hr and then reoxygenation for 1 hr. The addition of adenosine at 100 microM to the medium before hypoxia began significantly decreased enzyme leakage into medium during both hypoxia and reoxygenation. The adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), at the concentration of 100 microM, did not affect enzyme release, but the adenosine A2-receptor agonist 2-p-[2-car-boxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosi ne hydrochloride (CGS 21680) at the concentration of 100 nM, suppressed the injury caused by hypoxia and reoxygenation. There were decreases in cAMP contents and ATP levels in LLC-PK1 cells injured by hypoxia and reoxygenation. Adenosine (100 microM) restored ATP levels in the cells during reoxygenation. With adenosine, the intracellular cAMP level was increased prominently during reoxygenation. These results suggest that adenosine protects LLC-PK1 cells from injury caused by hypoxia and reoxygenation by increasing the intracellular cAMP level via adenosine A2 receptor.

  5. The A1 adenosine receptor as a new player in microglia physiology.

    PubMed

    Luongo, L; Guida, F; Imperatore, R; Napolitano, F; Gatta, L; Cristino, L; Giordano, C; Siniscalco, D; Di Marzo, V; Bellini, G; Petrelli, R; Cappellacci, L; Usiello, A; de Novellis, V; Rossi, F; Maione, S

    2014-01-01

    The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

  6. 'Domino' systems biology and the 'A' of ATP.

    PubMed

    Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V

    2013-01-01

    We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP.

  7. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.

  8. Rat cardiac myocyte adenosine transport and metabolism

    SciTech Connect

    Ford, D.A.; Rovetto, M.J.

    1987-01-01

    Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic fllux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations < 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable and nonsaturable processes. A minimum estimate of the V/sub max/ of myocytic adenosine kinase indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil. Extracellular adenosine taken up myocytes was rapidly phosphorylated to adenine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

  9. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    SciTech Connect

    Gruenhagen, Jason Alan

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  10. Calcium and ATP control multiple vital functions.

    PubMed

    Petersen, Ole H; Verkhratsky, Alexei

    2016-08-05

    Life on Planet Earth, as we know it, revolves around adenosine triphosphate (ATP) as a universal energy storing molecule. The metabolism of ATP requires a low cytosolic Ca(2+) concentration, and hence tethers these two molecules together. The exceedingly low cytosolic Ca(2+) concentration (which in all life forms is kept around 50-100 nM) forms the basis for a universal intracellular signalling system in which Ca(2+) acts as a second messenger. Maintenance of transmembrane Ca(2+) gradients, in turn, requires ATP-dependent Ca(2+) transport, thus further emphasizing the inseparable links between these two substances. Ca(2+) signalling controls the most fundamental processes in the living organism, from heartbeat and neurotransmission to cell energetics and secretion. The versatility and plasticity of Ca(2+) signalling relies on cell specific Ca(2+) signalling toolkits, remodelling of which underlies adaptive cellular responses. Alterations of these Ca(2+) signalling toolkits lead to aberrant Ca(2+) signalling which is fundamental for the pathophysiology of numerous diseases from acute pancreatitis to neurodegeneration. This paper introduces a theme issue on this topic, which arose from a Royal Society Theo Murphy scientific meeting held in March 2016.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

  11. Electrophysiological effects of ATP on brain neurones.

    PubMed

    Illes, P; Nieber, K; Nörenberg, W

    1996-12-01

    1. The electrophysiological effects of ATP on brain neurones are either due to the direct activation of P2 purinoceptors by the unmetabolized nucleotide or to the indirect activation of P1. purinoceptors by the degradation product adenosine. 2. Two subtypes of P2 purinoceptors are involved, a ligand-activated ion channel (P2X) and a G protein-coupled receptor (P2Y). Hence, the stimulation of P2X purinoceptors leads to a cationic conductance increase, while the stimulation of P2Y purinoceptors leads to a G protein-mediated opening or closure of potassium channels. 3. ATP may induce a calcium-dependent potassium current by increasing the intracellular Ca2+ concentration. This is due either to the entry of Ca2+ via P2X purinoceptors or to the activation of metabotropic P2Y purinoceptors followed by signaling via the G protein/phospholipase C/inositol 1,4,5-trisphosphate (IP3) cascade. Eventually, IP3 releases Ca2+ from its intracellular pools. 4. There is no convincing evidence for the presence of P2U purinoceptors sensitive to both ATP and UTP, or pyrimidinoceptors sensitive to UTP only, in the central nervous system (CNS). 5. ATP-sensitive P2X and P2Y purinoceptors show a wide distribution in the CNS and appear to regulate important neuronal functions.

  12. Extraction and analysis of adenosine triphosphate from aquatic environments

    USGS Publications Warehouse

    Stephens, Doyle W.; Shultz, David J.

    1981-01-01

    A variety of adenosine triphosphate (ATP) extraction procedures have been investigated for their applicability to samples from aquatic environments. The cold sulfuric-oxalic acid procedure was best suited to samples consisting of water, periphyton, and sediments. Due to cation and fulvic acid interferences, a spike with a known quantity of ATP was necessary to estimate losses when sediments were extracted. Variable colonization densities for periphyton required that several replicates be extracted to characterize accurately the periphyton community. Extracted samples were stable at room temperature for one to five hours, depending on the ATP concentration, if the pH was below 2. Neutralized samples which were quick frozen and stored at -30C were stable for months. (USGS)

  13. Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular.

    PubMed

    Bodin, P; Burnstock, G

    2001-12-01

    In response to increased shear stress, vascular endothelial cells release adenosine triphosphate (ATP) by an unknown mechanism. We have investigated this mechanism using different approaches. First, we discovered that quinacrine, used to locate intracellular stores of ATP bound to peptides, displayed a granular fluorescence, typical of vesicular storage. Second, we found that two inhibitors of vesicular transport (monensin and N-ethylmaleimide) produced a highly significant reduction in the release of ATP from vascular endothelial cells in response to increased shear stress. Preliminary experiments using inhibitors of the cystic fibrosis transmembrane regulator, the sulfonylurea receptor, and the multidrug resistance protein showed no involvement of these ATP-binding cassette transporter proteins (previously characterized in endothelial cells) in the mechanism of release of ATP. We suggest, therefore, that the release of ATP from vascular endothelial cells, like that of nerve cells, is probably by vesicular exocytosis.

  14. Fabrication of Adenosine Triphosphate-Molecule Recognition Chip by Means of Bioluminous Enzyme Luciferase

    NASA Astrophysics Data System (ADS)

    Tanii, Takashi; Goto, Tomomi; Iida, Tomoyuki; Koh-Masahara, Meishoku; Ohdomari, Iwao

    2001-10-01

    We have succeeded in detecting the adenosine triphosphate (ATP) concentration in a solution quantitatively using an ATP-molecule recognition chip. The ATP-molecule recognition chip is composed of a silicon photodiode on which bioluminous enzyme luciferase is immobilized. When the chip was immersed in an ATP-containing solution, the luciferase emitted light and the photoinduced current detected by the photodiode was in proportion to the ATP concentration. We found that the photoinduced current fits the Michaelis-Menten plot. These results indicate that the luciferase is successfully immobilized on the silicon chip without losing the bioluminous activity and that the proposed device enables us to detect the ATP concentration in a solution by measuring the photoinduced current.

  15. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    PubMed Central

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (–PO32−–Zr4+–) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP. PMID:27754349

  16. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    PubMed

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃(2-)-Zr(4+)-) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  17. ATP oscillations mediate inductive action of FGF and Shh signalling on prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2013-01-01

    Skeletal patterns are prefigured by prechondrogenic condensation. Morphogens such as fibroblast growth factor (FGF) and sonic hedgehog (Shh) specify the skeletal patterns in limb development. However, how morphogens regulate prechondrogenic condensation has remained unclear. Recently, it was demonstrated that synchronized Adenosine triphosphate (ATP) oscillations play a critical role in prechondrogenic condensation. Thus, the present study has focused on whether ATP oscillations mediate the actions of major developmental morphogens such as FGF and Shh on prechondrogenic condensation. It has been shown that both FGF and Shh signalling promoted cellular condensation but not chondrogenic differentiation and also induced ATP oscillations. In addition, blockage of FGF and Shh signalling prevented both ATP oscillations and prechondrogenic condensation. Furthermore, it was found that inhibition of ATP oscillations suppressed FGF/Shh-induced prechondrogenic condensation. These results indicate that ATP oscillations mediate the actions of FGF and Shh signalling on prechondrogenic condensation. This study proposes that morphogens organize skeletal patterns via ATP oscillations.

  18. Differential modulation by extracellular ATP of carotid chemosensory responses.

    PubMed

    Spergel, D; Lahiri, S

    1993-06-01

    The possibility that the carotid body has ATP surface receptors that mediate O2 chemoreception was tested. To distinguish between the event(s) initiating chemoreception and those at the neurotransmitter level, we also tested the chemosensory response to nicotine before and after ATP administration. Carotid bodies from cats anesthetized with pentobarbital sodium were perfused and superfused in vitro with modified Tyrode solution (PCO2 < 1 Torr, pH 7.4, 36 degrees C) equilibrated at PO2 > 400 or approximately 150 Torr while chemosensory discharge was recorded extracellularly. ATP and adenosine 5'-[gamma-thio]triphosphate stimulated discharge with similar dose dependence, whereas adenosine had little effect. ATP infusion for > or = 2 min evoked an initial stimulation of discharge followed by a decline to baseline (desensitization). Desensitization did not affect the response to hypoxia (perfusate flow interruption) but inhibited the response to nicotine (4-nmol pulse). Therefore, 1) the carotid body has surface ATP receptors that may mediate the chemosensory response to nicotine but not to hypoxia and 2) nicotinic receptors are not required for carotid body O2 chemoreception.

  19. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    NASA Astrophysics Data System (ADS)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  20. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent.

    PubMed

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-14

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg(2+) interacts with the highly charged ATP triphosphate group and Li(+) can co-bind with the native Mg(2+) to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg(2+) and Li(+) (i.e. which phosphate group(s) bind Mg(2+)/Li(+)) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg(2+)/Li(+) alone and combined: Mg(2+) prefers to bind ATP tridentately to each of the three phosphate groups, but Li(+) prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li(+) binds to Mg(2+)-loaded ATP. Hence, ATP-Mg-Li, like Mg(2+)-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  1. Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease

    PubMed Central

    Cha, Moon-Yong; Cho, Hyun Jin; Kim, Chaeyoung; Jung, Yang Ouk; Kang, Min Jueng; Murray, Melissa E.; Hong, Hyun Seok; Choi, Young-Joo; Choi, Heesun; Kim, Dong Kyu; Choi, Hyunjung; Kim, Jisoo; Dickson, Dennis W.; Song, Hyun Kyu; Cho, Jin Won; Yi, Eugene C.; Kim, Jungsu; Jin, Seok Min; Mook-Jung, Inhee

    2015-01-01

    Glycosylation with O-linked β-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5′-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aβ-treated cells. Indeed, Aβ bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aβ-induced impairment in ATP production and ATPase activity. These results indicate that Aβ-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aβ and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A. PMID:26358770

  2. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    PubMed Central

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-01-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways. PMID:28195155

  3. Phosphorylation potential and adenosine release during norepinephrine infusion in guinea pig heart

    SciTech Connect

    He, Miao-Xiang; Wangler, R.D.; Dillon, P.F.; Romig, G.D.; Sparks, H.V. )

    1987-11-01

    This study tested the hypothesis that adenosine released from isolated guinea pig hearts in response to norepinephrine is related to the cellular phosphorylation potential (PP;(ATP)/(ADP)(P{sub i})), where P{sub i} is inorganic phosphate. {sup 31}P-nuclear magnetic resonance (NMR) was used to measure the relative concentrations of P{sub i}, phosphocreatine (PCr), and ATP. After a control period, norepinephrine was infused for 20 min during which {sup 31}P-NMR spectra and samples of venous effluent were collected every minute. With norepinephrine infusion, PCr decreased rapidly to 72% of control by 8 min and then recovered to 80% of control for the remaining 12 min. ATP fell slowly to 70% of control over 20 min. P{sub i} increased to a peak at 2 min, then declined slowly to a steady state from 8 to 20 min. Adenosine release increased at 7 min and then slowly fell to a steady state from 10 to 20 min. There is hyperbolic relationship between adenosine release and PP; when the PP declines, a level is reached below which there is a rapid increase in adenosine release. These data support the hypothesis that adenosine release is regulated by the cellular PP as a closely related variable.

  4. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds

    PubMed Central

    Marín-Aguilar, Fabiola; Pavillard, Luis E.; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D.

    2017-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases. PMID:28146060

  5. Clickable 5'-γ-ferrocenyl adenosine triphosphate bioconjugates in kinase-catalyzed phosphorylations.

    PubMed

    Wang, Nan; She, Zhe; Lin, Yen-Chun; Martić, Sanela; Mann, David J; Kraatz, Heinz-Bernhard

    2015-03-23

    Clickable co-substrate: A tri-functional 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative containing a clickable site was synthesized. This compound is an effective co-substrate in kinase-catalyzed phosphorylation reactions, which can be detected by both electrochemical and immunoassay detection methods. The clickable reaction site makes direct modification possible, which greatly expands its application.

  6. The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

    NASA Technical Reports Server (NTRS)

    Bush, V. N.

    1973-01-01

    A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

  7. Dual effects of ATP on isolated arteries of the bovine eye.

    PubMed

    Ziganshina, Anna P; Ziganshin, Bulat A; Ziganshin, Ayrat U

    2012-08-01

    Although the presence of purinoreceptors has been shown in many human and animal arteries, there is few data yet about their role in the arteries of the eye. The purpose of the present study was to evaluate the effects of several agonists of purinoreceptors on isolated arteries of the bovine eye. Responses of isolated preparations of bovine ophthalmic (OA) and posterior ciliary arteries (PCA) to agonists of purinoreceptors (ATP, α,β-methylene-ATP-α,β-meATP, 2-methylthioATP-2meSATP, uridine-5'-triphosphate-UTP) as well as agonists of adreno-, cholino-, adenosine and histamine receptors were recorded by a standard organ bath method. ATP induced contractions of the intact vessels but caused relaxation of α,β-meATP-pretreated arteries. Contractile responses of PCA to high concentrations of ATP and α,β-meATP were significantly stronger than responses of OA, as well as relaxative responses to ATP and adenosine were significantly stronger in PCA than in OA. We suggest that there are several subtypes of functionally active purinoreceptors in both OA and PCA, although the potency of agonists of purinoreceptors to produce mechanical responses is higher in PCA than in OA. Purinoreceptors can be potential targets for new drugs, treating vascular pathology of the eye.

  8. ATP induces mild hypothermia in rats but has a strikingly detrimental impact on focal cerebral ischemia.

    PubMed

    Zhang, Meijuan; Li, Wenjin; Niu, Guangming; Leak, Rehana K; Chen, Jun; Zhang, Feng

    2013-01-01

    Ischemic stroke is a devastating condition lacking effective therapies. A promising approach to attenuate ischemic injury is mild hypothermia. Recent studies show that adenosine nucleotides can induce hypothermia in mice. The purpose of the present study was to test the hypothesis that adenosine 5'-triphosphate (ATP) induces mild hypothermia in rats and reduces ischemic brain injury. We found that intraperitoneal injections of ATP decreased core body temperature in a dose-dependent manner; the dose appropriate for mild hypothermia was 2 g/kg. When ATP-induced hypothermia was applied to stroke induced by middle cerebral artery occlusion, however, a neuroprotective effect was not observed. Instead, the infarct volume grew even larger in ATP-treated rats. This was accompanied by an increased rate of seizure events, hemorrhagic transformation, and higher mortality. Continuous monitoring of physiologic parameters revealed that ATP reduced heartbeat rate and blood pressure. ATP also increased blood glucose, accompanied by severe acidosis and hypocalcemia. Western blotting showed that ATP decreased levels of both phospho-Akt and total-Akt in the cortex. Our results reveal that, despite inducing hypothermia, ATP is not appropriate for protecting the brain against stroke. Instead, we show for the first time that ATP treatment is associated with exaggerated ischemic outcomes and dangerous systemic side effects.

  9. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  10. Additive Effectiveness Investigations in Alternative Fuels

    DTIC Science & Technology

    2014-05-01

    cetane value and storage stability for biodiesel . If only those properties were measured, it would have been deemed a success but a broader study...Paraffinic Kerosene ............................................................................. 2 2.1.2 FAME ( Biodiesel ...ASTM International, consensus standard organization ATP Adenosine TriPhosphate B20 Biodiesel , 20% blend with refined diesel (min) BOCLE Ball on

  11. [An adenosine triphosphate bioluminescence assay for detecting the number of living cells].

    PubMed

    Liu, S; Peng, Z; Wang, H; Lou, J; He, B; Tang, Q; Qiu, D

    2000-06-01

    The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.

  12. Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage.

    PubMed

    Björklund, Olga; Shang, Mingmei; Tonazzini, Ilaria; Daré, Elisabetta; Fredholm, Bertil B

    2008-10-31

    Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.

  13. An ATP gate controls tubulin binding by the tethered head of kinesin-1.

    PubMed

    Alonso, Maria C; Drummond, Douglas R; Kain, Susan; Hoeng, Julia; Amos, Linda; Cross, Robert A

    2007-04-06

    Kinesin-1 is a two-headed molecular motor that walks along microtubules, with each step gated by adenosine triphosphate (ATP) binding. Existing models for the gating mechanism propose a role for the microtubule lattice. We show that unpolymerized tubulin binds to kinesin-1, causing tubulin-activated release of adenosine diphosphate (ADP). With no added nucleotide, each kinesin-1 dimer binds one tubulin heterodimer. In adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable ATP analog, each kinesin-1 dimer binds two tubulin heterodimers. The data reveal an ATP gate that operates independently of the microtubule lattice, by ATP-dependent release of a steric or allosteric block on the tubulin binding site of the tethered kinesin-ADP head.

  14. Authentic role of ATP signaling in micturition reflex

    PubMed Central

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-01

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2−/− and P2X3−/− mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex. PMID:26795755

  15. Authentic role of ATP signaling in micturition reflex.

    PubMed

    Takezawa, Kentaro; Kondo, Makoto; Kiuchi, Hiroshi; Ueda, Norichika; Soda, Tetsuji; Fukuhara, Shinichiro; Takao, Tetsuya; Miyagawa, Yasushi; Tsujimura, Akira; Matsumoto-Miyai, Kazumasa; Ishida, Yusuke; Negoro, Hiromitsu; Ogawa, Osamu; Nonomura, Norio; Shimada, Shoichi

    2016-01-22

    Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2(-/-) and P2X3(-/-) mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex.

  16. Metabolic and functional consequences of inhibiting adenosine deaminase during renal ischemia in rats.

    PubMed Central

    Stromski, M E; van Waarde, A; Avison, M J; Thulin, G; Gaudio, K M; Kashgarian, M; Shulman, R G; Siegel, N J

    1988-01-01

    The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult. PMID:3263396

  17. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice.

    PubMed

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao

    2004-12-01

    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

  18. ATP-sulfurylase, sulfur-compounds, and plant stress tolerance

    PubMed Central

    Anjum, Naser A.; Gill, Ritu; Kaushik, Manjeri; Hasanuzzaman, Mirza; Pereira, Eduarda; Ahmad, Iqbal; Tuteja, Narendra; Gill, Sarvajeet S.

    2015-01-01

    Sulfur (S) stands fourth in the list of major plant nutrients after N, P, and K. Sulfate (SO42-), a form of soil-S taken up by plant roots is metabolically inert. As the first committed step of S-assimilation, ATP-sulfurylase (ATP-S) catalyzes SO42--activation and yields activated high-energy compound adenosine-5′-phosphosulfate that is reduced to sulfide (S2-) and incorporated into cysteine (Cys). In turn, Cys acts as a precursor or donor of reduced S for a range of S-compounds such as methionine (Met), glutathione (GSH), homo-GSH (h-GSH), and phytochelatins (PCs). Among S-compounds, GSH, h-GSH, and PCs are known for their involvement in plant tolerance to varied abiotic stresses, Cys is a major component of GSH, h-GSH, and PCs; whereas, several key stress-metabolites such as ethylene, are controlled by Met through its first metabolite S-adenosylmethionine. With the major aim of briefly highlighting S-compound-mediated role of ATP-S in plant stress tolerance, this paper: (a) overviews ATP-S structure/chemistry and occurrence, (b) appraises recent literature available on ATP-S roles and regulations, and underlying mechanisms in plant abiotic and biotic stress tolerance, (c) summarizes ATP-S-intrinsic regulation by major S-compounds, and (d) highlights major open-questions in the present context. Future research in the current direction can be devised based on the discussion outcomes. PMID:25904923

  19. Regulation of ClC-2 gating by intracellular ATP.

    PubMed

    Stölting, Gabriel; Teodorescu, Georgeta; Begemann, Birgit; Schubert, Julian; Nabbout, Rima; Toliat, Mohammad Reza; Sander, Thomas; Nürnberg, Peter; Lerche, Holger; Fahlke, Christoph

    2013-10-01

    ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.

  20. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

    PubMed

    Frieden, C; Patane, K

    1985-07-16

    The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

  1. Reexamination of magnetic isotope and field effects on adenosine triphosphate production by creatine kinase.

    PubMed

    Crotty, Darragh; Silkstone, Gary; Poddar, Soumya; Ranson, Richard; Prina-Mello, Adriele; Wilson, Michael T; Coey, J M D

    2012-01-31

    The influence of isotopically enriched magnesium on the creatine kinase catalyzed phosphorylation of adenosine diphosphate is examined in two independent series of experiments where adenosine triphosphate (ATP) concentrations were determined by a luciferase-linked luminescence end-point assay or a real-time spectrophotometric assay. No increase was observed between the rates of ATP production with natural Mg, (24)Mg, and (25)Mg, nor was any significant magnetic field effect observed in magnetic fields from 3 to 1,000 mT. Our results are in conflict with those reported by Buchachenko et al. [J Am Chem Soc 130:12868-12869 (2008)], and they challenge these authors' general claims that a large (two- to threefold) magnetic isotope effect is "universally observable" for ATP-producing enzymes [Her Russ Acad Sci 80:22-28 (2010)] and that "enzymatic phosphorylation is an ion-radical, electron-spin-selective process" [Proc Natl Acad Sci USA 101:10793-10796 (2005)].

  2. Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons.

    PubMed

    Flörchinger, Martin; Zimmermann, Marc; Traub, Michaela; Neuhaus, H Ekkehard; Möhlmann, Torsten

    2006-01-01

    In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.

  3. Adenosine, Ketogenic Diet and Epilepsy: The Emerging Therapeutic Relationship Between Metabolism and Brain Activity

    PubMed Central

    Masino, S.A; Kawamura, M; Wasser, C.D.; Pomeroy, L.T; Ruskin, D.N

    2009-01-01

    For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a “retaliatory metabolite.” As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor–based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed. PMID:20190967

  4. The interference of HEPES buffer during amperometric detection of ATP in clinical applications.

    PubMed

    Masson, Jean-Francois; Gauda, Estelle; Mizaikoff, Boris; Kranz, Christine

    2008-04-01

    HEPES-based biological buffer is subject to photooxidation upon exposure to fluorescent illumination. Thereby hydrogen peroxide is generated, which interferes with amperometric oxidoreductase-based biosensors for glucose or adenosine triphosphate (ATP). These biosensors operate at an oxidation potential above 500 mV vs. the standard calomel electrode (SCE) and involve hydrogen peroxide as the electroactive molecule detected at the electrode surface. False-positive detection of ATP was observed in HEPES buffer utilizing an amperometric microbiosensor based on the co-immobilization of glucose oxidase and hexokinase for detection of ATP in biological specimens. Electrochemical, mass spectrometric, (31)P NMR, and (1)H NMR studies indicate that complexation of ATP and HEPES induced by the presence of Ca(2+) in HEPES buffer decreases the photooxidation of HEPES. Consequently, the hydrogen peroxide background concentration is reduced, thereby leading to erroneous ATP detection at the dual-enzyme microbiosensor, which determines an increase in ATP via a reduced hydrogen peroxide signal.

  5. Fluorescent ATP analog mant-ATP reports dynein activity in the isolated Chlamydomonas axoneme

    NASA Astrophysics Data System (ADS)

    Feofilova, Maria; Howard, Jonathon

    Eukaryotic flagella are long rod-like extensions of cells, which play a fundamental role in single cell movement, as well as in fluid transport. Flagella contain a highly evolutionary conserved mechanical structure called the axoneme. The motion of the flagellum is generated by dynein motor proteins located all along the length of the axoneme. How the force production of motors is controlled spatially and temporally is still an open question. Therefore, monitoring dynein activity in the axonemal structure is expected to provide novel insights in regulation of the beat. We use high sensitivity fluorescence microscopy to monitor the binding and hydrolysis kinetics of the fluorescently labeled ATP analogue mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate), which is known to support dynein activity. By studying the kinetics of mant-ATP fluorescence, we identified distinct mant-ATP binding sites in the axoneme. The application of this method to axonemes with reduced amounts of dynein, showed evidence that one of the sites is associated with binding to dynein. In the future, we would like to use this method to find the spatial distribution of dynein activity in the axoneme.

  6. ATP sensitizes H460 lung carcinoma cells to cisplatin-induced apoptosis.

    PubMed

    Swennen, Els L R; Ummels, Vanessa; Buss, Irina; Jaehde, Ulrich; Bast, Aalt; Dagnelie, Pieter C

    2010-03-30

    Platinum resistance of cancer cells may evolve due to a decrease in intracellular drug accumulation, decreased cell permeability or by an increased deactivation of the drug by glutathione (GSH). The aim of this study was (1) to investigate the effect of adenosine 5'-triphosphate (ATP) on the cytotoxicity of cisplatin in a large cell lung carcinoma cell line (H460), and (2) to examine the potential involvement of increased cisplatin uptake, GSH depletion and pyrimidine starvation by ATP in this effect. H460 cells were harvested and seeded (5% CO(2); 37 degrees C). Subsequently, cells were incubated with medium or ATP followed by an incubation with cisplatin. Cytotoxicity screening was analyzed by the sulforhodamine B (SRB) colorimetric assay, lactate dehydrogenase and caspase-3/7 activity. Pre-incubation for 72h with 0.3 and 3mM ATP strongly enhanced the anti-proliferative potency of cisplatin 2.9- and 7.6-fold, respectively. Moreover, after incubation of H460 cells with 0.3mM ATP the intracellular platinum concentration increased, indicating increased cisplatin uptake by ATP. ATP, despite lowering the LD(50) of cisplatin, did not modulate GSH levels in H460 cells. ATP itself showed a biphasic effect on H460 cell growth: 0.3mM inhibited H460 cell growth via the pyrimidine starvation effect, activation of caspase-3/7 and LDH leakage, while 3mM ATP showed no effect on cell growth. In conclusion, ATP sensitizes the H460 cells to cisplatin-induced apoptosis. The effect of 0.3mM ATP is not due to GSH depletion but involves increased cisplatin uptake and pyrimidine starvation due to ATP conversion to adenosine followed by cellular uptake.

  7. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  8. Adenosine-Associated Delivery Systems

    PubMed Central

    Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

    2016-01-01

    Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

  9. Customized ATP towpreg

    NASA Astrophysics Data System (ADS)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placement from the robot head to the tool. This study examined the key towpreg ribbon properties and dimensions which play a significant role in ATP. Analysis of the heat transfer process window indicates that adequate heating can be achieved at lay down rates as high as 1 m/sec. While heat transfer did not appear to be the limiting factor, resin flow and fiber movement into tow lap gaps could be. Accordingly, consideration was given to towpreg ribbon having uniform yet non-rectangular cross sections. Dimensional integrity of the towpreg ribbon combined with customized ribbon architecture offer great promise for processing advances in ATP of high performance composites.

  10. A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

    PubMed Central

    Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

    2012-01-01

    In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

  11. Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

    PubMed Central

    Pani, Amar K.; Jiao, Yun; Sample, Kenneth J.; Smeyne, Richard J.

    2014-01-01

    Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models. PMID:24642754

  12. Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

    PubMed

    Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

    2014-03-01

    Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed.

  13. An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells.

    PubMed

    Chida, Junji; Yamane, Kazuhiko; Takei, Tunetomo; Kido, Hiroshi

    2012-05-21

    Firefly bioluminescence is widely used in the measurement of adenosine 5'-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin-luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris-EDTA-saturated phenol (phenol-TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol-TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

  14. Use of extractable adenosine triphosphate to estimate the viable cell mass in dental plaque samples obtained from monkeys.

    PubMed Central

    Robrish, S A; Kemp, C W; Bowen, W H

    1978-01-01

    The viable cell mass in plaque samples obtained from monkeys was estimated by determining the concentration of extractable adenosine triphosphate (ATP), and total cell mass was estimated by measuring the protein content. The results were expressed in terms of the specific ATP and protein contents of Streptococcus sanguis. The viable counts estimated by these techniques were comparable to or exceeded viable counts obtained by other investigators using conventional bacteriological methods. PMID:417674

  15. Optimization of adenosine 5'-triphosphate extraction for the measurement of acidogenic biomass utilizing whey wastewater.

    PubMed

    Lee, Changsoo; Kim, Jaai; Hwang, Seokhwan

    2006-08-01

    A set of experiments was carried out to maximize adenosine 5'-triphosphate (ATP) extraction efficiency from acidogenic culture using whey wastewater. ATP concentrations at different microbial concentrations increased linearly as microbial concentration decreased. More than 50% of ATP was extracted from the sample of 39 mg volatile suspended solids (VSS)/l compared to the sample of 2.8 g VSS/l. The ATP concentrations of the corresponding samples were 0.74+/-0.06 and 0.49+/-0.05 mg/l, respectively. For low VSS concentrations ranging from 39 to 92 mg/l, the extracted ATP concentration did not vary significantly at 0.73+/-0.01 mg ATP/l. Response surface methodology with a central composite in cube design for the experiments was used to locate the optimum for maximal ATP extraction with respect to boiling and bead beating treatments. The overall designed intervals were from 0 to 15 min and from 0 to 3 min for boiling and bead beating, respectively. The extracted ATP concentration ranged from 0.01 to 0.74 mg/l within the design boundary. The following is a partial cubic model where eta is the concentration of ATP and x ( k ) is the corresponding variable term (k=boiling time and bead beating time in order): eta=0.629+0.035x (1)-0.818x (2)-0.002x (1) x (2)-0.003x (1) (2) +0.254x (2) (2) +0.002x (1) (2) x (2). This model successfully approximates the response of ATP concentration with respect to the boiling- and bead beating-time. The condition for maximal ATP extraction was 5.6 min boiling without bead beating. The maximal ATP concentration using the model was 0.74 mg/l, which was identical to the experimental value at optimum condition for ATP extraction.

  16. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-10-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.

  17. Effect of adenosine triphosphate and some derivatives on cerebral blood flow and metabolism.

    PubMed Central

    Forrester, T; Harper, A M; MacKenzie, E T; Thomson, E M

    1979-01-01

    1. Responses of cerebral blood vessels to peri- and intravascular doses of ATP (adenosine triphosphate) and some derivatives were studied in cat and baboon. 2. Perivascular application of ATP to cat pial arterioles gave a threshold dilatory effect at a concentration of 10(-11) M. This figure is comparable to the amount of ATP calculated to be released from electrically stimulated brain slices. 3. It is concluded that adenine nucleotides have a major role to play in the local control of cerebral blood flow. 4. Intracarotid injection of ATP showed a calculated threshold effect at 4 x 10(8) M in the cat and 4 x 10(-9) M in the baboon. 5. The threshold response of the vasculature to intracarotid adenosine lay between 4 x 10(-7) M and 4 x 10(-6) M in the baboon. Little effect was produced with AMP, pyrophosphate and inorganic phosphate. 6. Intracarotid ATP increased the oxygen consumption of the baboon brain parenchyma. This effect was attributed in part to an elevation of the cellular cyclic AMP levels. 7. Osmotic disruption of the blood-brain barrier in baboon did not affect the vasodilatory or metabolic effect of intracarotid ATP. 8. It is postulated that circulating purine compounds mediate a form of metabolic communication inthe body. Also, release of purine compounds from active local nerves might influence cerebral blood flow. PMID:119042

  18. Modeling regulation of cardiac KATP and L-type Ca2+ currents by ATP, ADP, and Mg2+

    NASA Technical Reports Server (NTRS)

    Michailova, Anushka; Saucerman, Jeffrey; Belik, Mary Ellen; McCulloch, Andrew D.

    2005-01-01

    Changes in cytosolic free Mg(2+) and adenosine nucleotide phosphates affect cardiac excitability and contractility. To investigate how modulation by Mg(2+), ATP, and ADP of K(ATP) and L-type Ca(2+) channels influences excitation-contraction coupling, we incorporated equations for intracellular ATP and MgADP regulation of the K(ATP) current and MgATP regulation of the L-type Ca(2+) current in an ionic-metabolic model of the canine ventricular myocyte. The new model: 1), quantitatively reproduces a dose-response relationship for the effects of changes in ATP on K(ATP) current, 2), simulates effects of ADP in modulating ATP sensitivity of K(ATP) channel, 3), predicts activation of Ca(2+) current during rapid increase in MgATP, and 4), demonstrates that decreased ATP/ADP ratio with normal total Mg(2+) or increased free Mg(2+) with normal ATP and ADP activate K(ATP) current, shorten action potential, and alter ionic currents and intracellular Ca(2+) signals. The model predictions are in agreement with experimental data measured under normal and a variety of pathological conditions.

  19. Cell death of bioenergetically compromised and transcriptionally challenged CLL lymphocytes by chlorinated ATP

    PubMed Central

    Balakrishnan, Kumudha; Stellrecht, Christine M.; Genini, Davide; Ayres, Mary; Wierda, William G.; Keating, Michael J.; Leoni, Lorenzo M.; Gandhi, Varsha

    2005-01-01

    Myeloid cell leukemia-1 (MCL-1) acts as a key survival factor for chronic lymphocytic leukemia (CLL) cells. In addition, dissipation of cellular bioenergy may impose a lethal effect on these quiescent cells. Previously, in multiple myeloma cell lines we demonstrated that halogenated adenosine (8-Cl-Ado) was phosphorylated to triphosphate (8-Cl–adenosine triphosphate [ATP]), which preferentially incorporated into mRNA and inhibited RNA synthesis by premature transcription termination. Furthermore, 8-Cl-ATP accumulation was associated with a decline in cellular bioenergy. Based on these actions, we hypothesized that 8-Cl-Ado would be ideal to target CLL lymphocytes. In the present study we demonstrate that leukemic lymphocytes incubated with 8-Cl-Ado display time- and dose-dependent increase in the accumulation of 8-Cl-ATP, with a parallel depletion of the endogenous ATP pool. Inhibition of global RNA synthesis resulted in a significant decline in the expression of transcripts with a short half-life such as MCL1. Consistent to this, protein expression of MCL-1 but not B-cell lymphoma–2 (BCL-2) was decreased. Furthermore, 8-Cl-ATP induced programmed cell death, as suggested by caspases activation, cleavage of caspase 3, and PARP (poly–adenosine diphosphate [ADP]–ribose polymerase), and increased DNA fragmentation. In conclusion, 8-Cl-Ado induces apoptosis in CLL lymphocytes by targeting cellular bioenergy as well as RNA transcription and translation of key survival genes such as MCL1. PMID:15718423

  20. Adenosine Generated in the Bone Marrow Niche Through a CD38-Mediated Pathway Correlates With Progression of Human Myeloma

    PubMed Central

    Horenstein, Alberto L; Quarona, Valeria; Toscani, Denise; Costa, Federica; Chillemi, Antonella; Pistoia, Vito; Giuliani, Nicola; Malavasi, Fabio

    2016-01-01

    Human myeloma cells express CD38 at high levels and grow in hypoxic niches inside the bone marrow. Myeloma cells respond to hypoxia with metabolic changes leading to aerobic glycolysis, thus reducing adenosine triphosphate (ATP) and increasing NAD+. Our hypothesis is that these conditions favor the enzymatic pathways involved in the production of adenosine in the niche. Within the niche, NAD+ is able to activate a discontinuous adenosinergic pathway that relies upon CD38, CD203a and CD73 or TRACP, according to the environmental pH. The observed variability in adenosine concentrations in bone marrow aspirates is a result of the interactions taking place among myeloma and other cells in the bone marrow niche. A pilot study showed that adenosine profiles differ during disease progression. Adenosine levels were significantly higher in the bone marrow plasma of patients with symptomatic myeloma and correlated with ISS staging, suggesting that adenosine is produced in the myeloma niche at micromolar levels by an ectoenzymatic network centered on CD38. Adenosine levels increase with disease aggressiveness, a finding that supports adenosine as a potential marker of myeloma progression. PMID:27761584

  1. Adenosine receptor targets for pain.

    PubMed

    Sawynok, J

    2016-12-03

    The main focus for the development of adenosine targets as analgesics to date has been A1Rs due to its antinociceptive profile in various preclinical pain models. The usefulness of systemic A1R agonists may be limited by other effects (cardiovascular, motor), but enhanced selectivity for pain might occur with partial agonists, potent and highly selective agonists, or allosteric modulators. A2AR agonists exhibit some peripheral pronociceptive effects, but also act on immune cells to suppress inflammation and on spinal glia to suppress pain signaling and may be useful for inflammatory and neuropathic pain. A2BR agonists exhibit peripheral proinflammatory effects on immune cells, but also spinal antinociceptive effects similar to A2AR agonists. A3Rs are now demonstrated to produce antinociception in several preclinical neuropathic pain models, with mechanistic actions on glial cells, and may be useful for neuropathic pain. Endogenous adenosine levels can be augmented by inhibition of metabolism (via adenosine kinase) or increased generation (via nucleotidases), and these approaches have implications for pain. Endogenous adenosine contributes to antinociception by several pharmacological agents, herbal remedies, acupuncture, transcutaneous electrical nerve stimulation, exercise, joint mobilization, and water immersion via spinal and/or peripheral effects, such that this system appears to constitute a major pain regulatory system. Finally, caffeine inhibits A1-, A2A- and A3Rs with similar potency, and dietary caffeine intake will need attention in trials of: (a) agonists and/or modulators acting at these receptors, (b) some pharmacological and herbal analgesics, and (c) manipulations that enhance endogenous adenosine levels, all of which are inhibited by caffeine and/or A1R antagonists in preclinical studies. All adenosine receptors have effects on spinal glial cells in regulating nociception, and gender differences in the involvement of such cells in chronic

  2. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  3. How Reliable Are ATP Bioluminescence Meters in Assessing Decontamination of Environmental Surfaces in Healthcare Settings?

    PubMed Central

    Omidbakhsh, Navid; Ahmadpour, Faraz; Kenny, Nicole

    2014-01-01

    Background Meters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry? Objective This study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals’ interference in their readings. Methods First, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter’s ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings. Results All four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated. Conclusions Apart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries. PMID:24940751

  4. Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

    PubMed Central

    Fuentes, Eduardo; Pereira, Jaime; Mezzano, Diego; Alarcón, Marcelo; Caballero, Julio; Palomo, Iván

    2014-01-01

    Background The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. Objectives The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. Results Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. Conclusion Therefore

  5. ROLE OF ATP IN REGULATING RENAL MICROVASCULAR FUNCTION AND IN HYPERTENSION

    PubMed Central

    Guan, Zhengrong; Inscho, Edward W.

    2011-01-01

    Adenosine triphosphate (ATP) is an essential energy substrate for cellular metabolism but it can also influence many biological processes when released into the extracellular milieu. Research has established that extracellular ATP acts as an autocrine/paracrine factor that regulates many physiological functions. Alternatively, excessive extracellular ATP levels contribute to pathophysiological processes such as inflammation, cell proliferation and apoptosis, and atherosclerosis. Renal P2 receptors are widely distributed throughout glomeruli, vasculature and tubular segments, and participate in controlling renal vascular resistance, mediating renal autoregulation, and regulating tubular transport function. This review will focus on the role of ATP-P2 receptor signaling in regulating renal microvascular function and autoregulation, recent advances on the role of ATP-P2 signaling in hypertension-associated renal vascular injury, and emerging new directions. PMID:21768526

  6. Sensitivity of small myosin II ensembles from different isoforms to mechanical load and ATP concentration

    NASA Astrophysics Data System (ADS)

    Erdmann, Thorsten; Bartelheimer, Kathrin; Schwarz, Ulrich S.

    2016-11-01

    Based on a detailed crossbridge model for individual myosin II motors, we systematically study the influence of mechanical load and adenosine triphosphate (ATP) concentration on small myosin II ensembles made from different isoforms. For skeletal and smooth muscle myosin II, which are often used in actomyosin gels that reconstitute cell contractility, fast forward movement is restricted to a small region of phase space with low mechanical load and high ATP concentration, which is also characterized by frequent ensemble detachment. At high load, these ensembles are stalled or move backwards, but forward motion can be restored by decreasing ATP concentration. In contrast, small ensembles of nonmuscle myosin II isoforms, which are found in the cytoskeleton of nonmuscle cells, are hardly affected by ATP concentration due to the slow kinetics of the bound states. For all isoforms, the thermodynamic efficiency of ensemble movement increases with decreasing ATP concentration, but this effect is weaker for the nonmuscle myosin II isoforms.

  7. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

  8. Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

    PubMed

    Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

    2014-11-01

    Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay.

  9. Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates†

    PubMed Central

    Fontes, Rui; Günther Sillero, Maria A.; Sillero, Antonio

    1998-01-01

    Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p4A) and adenosine 5′-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), and adenosine(5′)pentaphospho(5′)adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The Km values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates). PMID:9620965

  10. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    NASA Astrophysics Data System (ADS)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  11. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods.

    PubMed

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-08

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ~56 nm and diameter ~12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  12. Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells.

    PubMed

    Cutarelli, Alessandro; Marini, Mario; Tancredi, Virginia; D'Arcangelo, Giovanna; Murdocca, Michela; Frank, Claudio; Tarantino, Umberto

    2016-05-01

    In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells.

  13. Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

    PubMed

    Zeng, Xiaodan; Zhang, Xiaoling; Yang, Wen; Jia, Hongying; Li, Yamin

    2012-05-01

    An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

  14. Detection of adenosine 5'-triphosphate by fluorescence variation of oligonucleotide-templated silver nanoclusters.

    PubMed

    Lee, Jennifer Daneen; Cang, Jinshun; Chen, Ying-Chieh; Chen, Wei-Yu; Ou, Chung-Mao; Chang, Huan-Tsung

    2014-08-15

    Oligonucleotide-templated Ag nanoclusters (DNA-Ag NCs) prepared from AgNO3 using an oligonucleotide (5'-TAACCCCTAACCCCT-3') as a template and NaBH4 as a reducing agent have been used for sensing of adenosine 5'-triphosphate (ATP). The fluorescence intensity and emission wavelength of DNA-Ag NCs are dependent on the pH value and ATP concentration. At pH 3.0 and 11.0, ATP shows greater effects on fluorescence of the DNA-Ag NCs. Upon increasing ATP concentration from 10 to 50μM, their emission wavelength at pH 3.0 shifts from 525 to 585nm. At pH 11.0, their fluorescence intensity (510nm) increases upon increasing ATP concentration. The circular dichroism (CD), electrospray ionization-mass spectrometry (ESI-MS), absorption, and fluorescence results indicate that ATP and pH affect the interactions between DNAs and Ag atoms, resulting in changes in their fluorescence. The DNA-Ag NCs allow detection of ATP over a concentration range of 0.1-10μM, with a limit of detection 33nM. Practicality of the DNA-Ag NCs probe has been validated with the determination of ATP concentrations in the lysate of MDA-MB-231 breast carcinoma cells.

  15. Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate.

    PubMed

    Song, Weiling; Zhang, Qiao; Xie, Xuxu; Zhang, Shusheng

    2014-11-15

    In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6 × 10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

  16. Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

    PubMed

    Hacker, Stephan M; Welter, Moritz; Marx, Andreas

    2015-03-09

    This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

  17. Extracellular ATP metabolism on vascular endothelial cells: A pathway with pro-thrombotic and anti-thrombotic molecules.

    PubMed

    Fuentes, Eduardo; Palomo, Iván

    2015-12-01

    Vascular endothelial contributes to the metabolism and interconversion of extracellular adenine nucleotides via ecto-ATPase/ADPase (CD39) and ecto-5'nucleotidase (CD73) activities. These enzymes collectively dephosphorylate ATP, ADP, and AMP with the production of additional adenosine. In the vascular system, adenine nucleotides (ATP and ADP) and nucleoside adenosine represent an important class of extracellular molecules involved in modulating the processes linked to vascular thrombosis exerting various effects in platelets. Yet, the mechanisms by which the extracellular ATP metabolism in the local environment trigger pro-thrombotic and anti-thrombotic states are yet to be fully elucidated. In this article, the relative contribution of extracellular ATP metabolism in platelet regulation is explored.

  18. A novel aptasensor for the ultra-sensitive detection of adenosine triphosphate via aptamer/quantum dot based resonance energy transfer.

    PubMed

    Li, Zheng; Wang, Yijing; Liu, Ying; Zeng, Yongyi; Huang, Aimin; Peng, Niancai; Liu, Xiaolong; Liu, Jingfeng

    2013-09-07

    We designed a novel aptamer based biosensor (aptasensor) for ultrasensitive detection of adenosine triphosphate (ATP) through resonance energy transfer (RET). The ATP aptamer was modified with Cy3 at the 3' end, and a green quantum dot (525) was attached to the 5' end of its complementary sequence respectively. The ATP aptamer and its complementary sequence could assemble into a duplex structure in the absence of target ATP, and then decrease the distance between the quantum dot and Cy3 which could produce significant RET signal. Upon ATP binding, the ATP aptamer could dissociate with its complementary sequence and then increase the distance between the quantum dot and Cy3 which would significantly decrease the RET signal. Therefore, the ATP detection could be easily achieved through detection of the fluorescence intensity ratio between 525 nm and 560 nm. The results show that the emission fluorescence intensity ratio of 525/560 is linearly related to the logarithmic concentration of ATP. The linear range of this aptasensor is from 0.1 nM to 1 μM, and the detection limit is lower down to 0.01 nM. Excellent selectivity of this aptasensor for ATP has been demonstrated through the detection of thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and adenosine diphosphate (ADP) respectively as control. The method we described here could easily detect ATP with excellent selectivity, linearity and sensitivity down to the nanomolar range, as well as avoid photobleaching.

  19. Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging.

    PubMed

    Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

    2015-05-07

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.

  20. Effects of caffeine on fractional flow reserve values measured using intravenous adenosine triphosphate.

    PubMed

    Nakayama, Masafumi; Chikamori, Taishiro; Uchiyama, Takashi; Kimura, Yo; Hijikata, Nobuhiro; Ito, Ryosuke; Yuhara, Mikio; Sato, Hideaki; Kobori, Yuichi; Yamashina, Akira

    2017-01-21

    We investigated the effects of caffeine intake on fractional flow reserve (FFR) values measured using intravenous adenosine triphosphate (ATP) before cardiac catheterization. Caffeine is a competitive antagonist for adenosine receptors; however, it is unclear whether this antagonism affects FFR values. Patients were evenly randomized into 2 groups preceding the FFR study. In the caffeine group (n = 15), participants were given coffee containing 222 mg of caffeine 2 h before the catheterization. In the non-caffeine group (n = 15), participants were instructed not to take any caffeine-containing drinks or foods for at least 12 h before the catheterization. FFR was performed in patients with more than intermediate coronary stenosis using the intravenous infusion of ATP at 140 μg/kg/min (normal dose) and 170 μg/kg/min (high dose), and the intracoronary infusion of papaverine. FFR was followed for 30 s after maximal hyperemia. In the non-caffeine group, the FFR values measured with ATP infusion were not significantly different from those measured with papaverine infusion. However, in the caffeine group, the FFR values were significantly higher after ATP infusion than after papaverine infusion (P = 0.002 and P = 0.007, at normal and high dose ATP vs. papaverine, respectively). FFR values with ATP infusion were significantly increased 30 s after maximal hyperemia (P = 0.001 and P < 0.001 for normal and high dose ATP, respectively). The stability of the FFR values using papaverine showed no significant difference between the 2 groups. Caffeine intake before the FFR study affected FFR values and their stability. These effects could not be reversed by an increased ATP dose.

  1. Nucleotide binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe.

    PubMed

    Cheng, K; Koland, J G

    1996-01-05

    The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.

  2. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

    SciTech Connect

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-08-31

    Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

  3. Mechanisms of ATP release and signalling in the blood vessel wall

    PubMed Central

    Lohman, Alexander W.; Billaud, Marie; Isakson, Brant E.

    2012-01-01

    The nucleotide adenosine 5′-triphosphate (ATP) has classically been considered the cell's primary energy currency. Importantly, a novel role for ATP as an extracellular autocrine and/or paracrine signalling molecule has evolved over the past century and extensive work has been conducted to characterize the ATP-sensitive purinergic receptors expressed on almost all cell types in the body. Extracellular ATP elicits potent effects on vascular cells to regulate blood vessel tone but can also be involved in vascular pathologies such as atherosclerosis. While the effects of purinergic signalling in the vasculature have been well documented, the mechanism(s) mediating the regulated release of ATP from cells in the blood vessel wall and circulation are now a key target of investigation. The aim of this review is to examine the current proposed mechanisms of ATP release from vascular cells, with a special emphasis on the transporters and channels involved in ATP release from vascular smooth muscle cells, endothelial cells, circulating red blood cells, and perivascular sympathetic nerves, including vesicular exocytosis, plasma membrane F1/F0-ATP synthase, ATP-binding cassette (ABC) transporters, connexin hemichannels, and pannexin channels. PMID:22678409

  4. ATP as a multi-target danger signal in the brain

    PubMed Central

    Rodrigues, Ricardo J.; Tomé, Angelo R.; Cunha, Rodrigo A.

    2015-01-01

    ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, in astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R) as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR), which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection. PMID:25972780

  5. Critical role of ATP-induced ATP release for Ca2+ signaling in nonsensory cell networks of the developing cochlea

    PubMed Central

    Ceriani, Federico; Pozzan, Tullio; Mammano, Fabio

    2016-01-01

    Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 μm⋅s−1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems. PMID:27807138

  6. Adenosine-activated potassium current in smooth muscle cells isolated from the pig coronary artery.

    PubMed Central

    Dart, C; Standen, N B

    1993-01-01

    1. The perforated patch technique with nystatin or amphotericin was used to record whole cell currents activated by adenosine in smooth muscle cells isolated enzymatically from pig coronary arteries. 2. Adenosine (5-40 microM) activated an outward current at a holding potential of 0 mV in 5 mM [K+]o and an inward current at -60 mV in 143 mM [K+]o. The dependence of the reversal potential for the adenosine-activated current on [K+]o suggests that it flows through K+ channels, while its current-voltage relation is consistent with the channels showing little voltage dependence. 3. The adenosine-activated current was inhibited by the sulphonylurea glibenclamide (5 microM) and by phencyclidine (5 microM). It was unaffected by charybdotoxin (50 nM) or apamin (100 nM), blockers of large and small conductance Ca(2+)-activated K+ channels respectively. 4. At -60 mV in 143 mM K+ solution, openings of single channels passing a current of just over -2 pA could sometimes be detected in the absence of adenosine. Openings became more frequent after the application of adenosine, with several levels then being detected. Openings of channels with a larger conductance were sometimes also seen in the presence of adenosine. Fluctuation analysis gave somewhat lower estimates of unitary current than did direct measurements. 5. The effect of adenosine could be mimicked by the A1 receptor agonist CCPA (2-chloro-N6-cyclopentyladenosine), while the A2 agonist CGS 21680 (2-p-(2-carboxethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride) was without effect. The response to adenosine was inhibited by the A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), but was unaffected by the A2 antagonist CGS 15943A (5-amino-9-chloro-2-(2-furanyl)-1,2,4- triazolo[1,5-C]quinazoline monomethanesulphonate). 6. Our results suggest that adenosine acts at an A1 receptor to activate K+ channels. We consider it most likely that these are ATP-dependent K+ channels. We discuss the mechanism by

  7. Curtains for ATP?

    NASA Astrophysics Data System (ADS)

    The administration's efforts to keep various technology-transfer programs afloat in the budget process appear to be stalled. House Science Committee chair Robert Walker (R-Pa.) advised in early April that the Republican agenda for the pending budget process entails zeroing out the Commerce Department's Advanced Technology Program (ATP), which was funded at 431 million in fiscal year 1995. The ATP would lose about 90 million from its FY 95 budget. Although Walker says that the Republican leadership has no intention to dictate to the subcommittees how cuts should be made, they will be held to the "fairly severe caps" established by the House Budget Committee. In other words, Walker says, if ATP stays, something else will have to go in its place. In addition, a bill to rescind about 223 million from the FY 1995 budget of the Technology Reinvestment Project and another 77 million from TRP's FY 1994 budget, which has not been spent, is heading for the president's signature. Yet Walker says while he supports the merits of technology transfer, "the question is do you have to create government programs to get the technology out?"

  8. A target-triggered strand displacement reaction cycle: the design and application in adenosine triphosphate sensing.

    PubMed

    Cheng, Sheng; Zheng, Bin; Wang, Mozhen; Lam, Michael Hon-Wah; Ge, Xuewu

    2014-02-01

    A strand displacement reaction (SDR) system that runs solely on oligonucleotides has been developed for the amplification detection of adenosine triphosphate (ATP). It involves a target-induced SDR and an entropy-driven catalytic cycle of two SDRs with five oligonucleotides, denoted as substrate, fuel, catalyst, C-1, and C-2. Catalyst, released from the ATP aptamer-catalyst duplex by ATP molecule, catalyzes the SDRs to finally form the substrate-fuel duplex. All of the intermediates in the catalytic SDR processes have been identified by polyacrylamide gel electrophoresis (PAGE) analysis. The introduction of ATP into the SDR system will induce the ATP aptamer to form G-quadruplex conformation so as to release catalyst and trigger the SDR cycle. When the substrate and C-2 oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibited a "turn-on" response for ATP. The condition for detecting ATP, such as Mg²⁺ concentration, has been optimized to afford a detection limit of 20 nM. This work provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing.

  9. Intracellular Adenosine Triphosphate Deprivation through Lanthanide-Doped Nanoparticles.

    PubMed

    Tian, Jing; Zeng, Xiao; Xie, Xiaoji; Han, Sanyang; Liew, Oi-Wah; Chen, Yei-Tsung; Wang, Lianhui; Liu, Xiaogang

    2015-05-27

    Growing interest in lanthanide-doped nanoparticles for biological and medical uses has brought particular attention to their safety concerns. However, the intrinsic toxicity of this new class of optical nanomaterials in biological systems has not been fully evaluated. In this work, we systematically evaluate the long-term cytotoxicity of lanthanide-doped nanoparticles (NaGdF4 and NaYF4) to HeLa cells by monitoring cell viability (mitochondrial activity), adenosine triphosphate (ATP) level, and cell membrane integrity (lactate dehydrogenase release), respectively. Importantly, we find that ligand-free lanthanide-doped nanoparticles induce intracellular ATP deprivation of HeLa cells, resulting in a significant decrease in cell viability after exposure for 7 days. We attribute the particle-induced cell death to two distinct cell death pathways, autophagy and apoptosis, which are primarily mediated via the interaction between the nanoparticle and the phosphate group of cellular ATP. The understanding gained from the investigation of cytotoxicity associated with lanthanide-doped nanoparticles provides keen insights into the safe use of these nanoparticles in biological systems.

  10. Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.

    PubMed

    Zhao, Qiang; Lv, Qin; Wang, Hailin

    2015-08-15

    We previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets.

  11. Antihyperlipidemic activity of adenosine triphosphate in rabbits fed a high-fat diet and hyperlipidemic patients.

    PubMed

    Zhang, Lianshan; Liang, Libin; Tong, Tong; Qin, Yuguo; Xu, Yanping; Tong, Xinglong

    2016-10-01

    Context Recently, adenosine triphosphate (ATP) was occasionally found to decrease the triglyceride (TG) levels in several hyperlipidemic patients in our clinical practice. Objective The study investigates the anti-hyperlipidemic effects of ATP in a high-fat fed rabbit model and hyperlipidemic patients. Materials and methods Twenty-four rabbits were randomly divided into three groups of eight animals each as follows: normal diet, high-fat diet and high-fat diet + ATP group. ATP supplementation (40 mg/day) was started at the 20th day and lasted for 10 days. Serum concentrations of total cholesterol (TC), TG, LDL-C, HDL-C were measured on the 20th day and 30th day. Heart, liver and aorta were subjected histopathological examination. Twenty outpatients diagnosed primary hyperlipidemia took ATP at a dose of 60 mg twice a day for 1 week. Results Feeding rabbits with a high-fat diet resulted in a significant elevation of lipid parameters including TC, TG, LDL-C, VLDL-C compared to the normal diet group (p < 0.01). ATP treatment significantly decreased serum TG level (p < 0.01), whilst other parameters remained statistically unaltered. Meanwhile, ATP significantly reduced the thickness of fat layer in cardiac epicardium (p < 0.05) and pathological gradation of ballooning degeneration in hepatocytes (p < 0.05). After taking ATP for 1 week, hyperlipidemia patients exhibited a significant decrease of TG (p < 0.01), but other lipid parameters had no significant change. Discussion and conclusion The study indicates that ATP selectively decreases serum TG levels in high-fat diet rabbits and hyperlipidemic patients. Therefore, ATP supplementation may provide an effective approach to control TG level.

  12. Pharmacological dissociation of UTP- and ATP-elicited contractions and relaxations in isolated rat aorta.

    PubMed

    Garcia-Velasco, G; Sanchez, M; Hidalgo, A; Garcia de Boto, M J

    1995-12-29

    Effects of UTP have been described in many tissues, but it is not clear whether these are due to purinoceptors. Specific receptors for UTP, 'pyrimidinoceptors', and 'nucleotide receptors' have also been proposed. We pharmacologically characterized the receptors involved in the ATP- and UTP-induced contraction under basal tone and the relaxation of raised tone elicited by noradrenaline in isolated rat aorta. The rank order of potency for the agonists for the contraction was alpha,beta-methylene ATP > > ATP, and the desensitization by alpha,beta-methylene ATP suggests that ATP contractions were mediated via P2X purinoceptors which were located on the vascular smooth muscle. The rank order of potency of the agonists for relaxation was 2-methyl-thio ATP > > ATP, which is suggestive of a P2Y purinoceptor. However, the relaxation seems to be unrelated to the classical P2Y subtype and a heterogeneous population of purinoceptors might therefore exist. The evidence comes from the distinct location and the different pharmacological effect of reactive blue 2 on 2-methyl-thio ATP and ATP receptors. 2-Methyl-thio ATP produced an endothelium-dependent relaxation while ATP-induced relaxation was produced via endothelium-dependent and endothelium-independent mechanisms, unrelated to adenosine receptors. It is unlikely that UTP-induced contractions and the endothelium-dependent relaxation were produced via purinoceptors since the pharmacology is not consistent with that of the classical P2 purinoceptors studied. Furthermore, UTP-sensitive receptors showed a pharmacological property that was also distinct from that of the 'nucleotide' or P2U receptor reported. The results suggest the presence of a heterogeneous population of purinoceptors and pyrimidinoceptors pharmacologically different from the receptors for ATP.

  13. Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

    PubMed Central

    Lipitz, Jeffrey B.; Dahl, Gerhard

    2010-01-01

    Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury. PMID:20876360

  14. Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

    PubMed Central

    Newby, A C

    1980-01-01

    1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells. PMID:6249264

  15. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  16. IV ATP potentiates midazolam sedation as assessed by bispectral index.

    PubMed

    Sakurai, Satoru; Fukunaga, Atsuo; Ichinohe, Tatsuya; Kaneko, Yuzuru

    2014-01-01

    In this study, by measuring bispectral index (BIS), we tested the hypothesis that intravenous adenosine 5'-triphosphate (ATP) infusion would deepen the level of midazolam-induced sedation. Ten healthy volunteers underwent 2 experiments with at least 2 weeks' interval: immediately after intravenous bolus administration of midazolam (0.04 mg/kg), they received continuous infusion of either ATP infusion (100 μg/kg/min) or placebo (saline) for 40 minutes in a double-blind, randomized, crossover manner. Changes in BIS values and responsiveness to verbal command as well as cardiorespiratory variables were observed throughout the study periods. Administration of midazolam alone reduced BIS value from control: 97 ± 1 to 68 ± 18 at 25 minutes, which was accompanied by significant cardiopulmonary depressant effects, while maintaining responsiveness to verbal command (consciousness) throughout the study period. Coadministration of ATP with midazolam further reduced BIS value to 51 ± 13, associated with complete loss of consciousness without adverse effect on the cardiorespiratory systems. We conclude that the addition of ATP infusion to midazolam significantly enhances midazolam sedation without disturbing cardiorespiratory functions.

  17. Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation*

    PubMed Central

    Herrmann, Jonathan; Ravilious, Geoffrey E.; McKinney, Samuel E.; Westfall, Corey S.; Lee, Soon Goo; Baraniecka, Patrycja; Giovannetti, Marco; Kopriva, Stanislav; Krishnan, Hari B.; Jez, Joseph M.

    2014-01-01

    Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5′-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3′-phosphoadenosine 5′-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway. PMID:24584934

  18. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    NASA Astrophysics Data System (ADS)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

  19. MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

    PubMed

    Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

    2010-04-01

    Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

  20. Wound-induced ATP release and EGF receptor activation in epithelial cells

    PubMed Central

    Yin, Jia; Xu, Keping; Zhang, Jing; Kumar, Ashok; Yu, Fu-Shin X.

    2007-01-01

    Summary We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-γ-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-γ-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-γ-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing. PMID:17284517

  1. [A review of recent researches on correlation between ATP and acupuncture efficacies].

    PubMed

    Chen, Bo; Guo, Yi; Zhao, Xue; Liu, Yang-Yang; Li, Zhong-Zheng; Li, Ying-Hong; Guo, Yong-Ming

    2012-08-01

    It has been documented that adenosine triphosphate (ATP) is a multifunctional nucleoside triphosphate used in cells, including chemical energy transportation, extra- and intracellular signaling, cell structure maintaining, DNA and RNA synthesis, etc. In the present paper, the authors reviewed studies on the involvement of ATP in different efficacies of acupuncture intervention from the following four aspects. 1) ATP release in the stimulated acupoint area is one of the key factors for producing acupuncture analgesia; 2) Acupuncture induced suppression of ATP activity in the central nervous system results in pain relief; 3) ATP application on the human body surface may strengthen the sensation propagation along the meridian; 4) Favorable regulation of acupuncture intervention on the abnormal functional activities of some viscera often accompanies with an increase of ATP content and ATPase activity in the related internal organs. It has been proposed that ATP, Ca2+ and reactive oxygen species (ROS) are closely related each other in the life activities of the organism. Hence, a reasonable regulation on ATP levels in the related organs of the body may be a new approach for raising clinical therapeutic effects of acupuncture therapy.

  2. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects.

    PubMed

    Kojima, Shuji; Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells.

  3. Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

    PubMed

    Séror, Claire; Melki, Marie-Thérèse; Subra, Frédéric; Raza, Syed Qasim; Bras, Marlène; Saïdi, Héla; Nardacci, Roberta; Voisin, Laurent; Paoletti, Audrey; Law, Frédéric; Martins, Isabelle; Amendola, Alessandra; Abdul-Sater, Ali A; Ciccosanti, Fabiola; Delelis, Olivier; Niedergang, Florence; Thierry, Sylvain; Said-Sadier, Najwane; Lamaze, Christophe; Métivier, Didier; Estaquier, Jérome; Fimia, Gian Maria; Falasca, Laura; Casetti, Rita; Modjtahedi, Nazanine; Kanellopoulos, Jean; Mouscadet, Jean-François; Ojcius, David M; Piacentini, Mauro; Gougeon, Marie-Lise; Kroemer, Guido; Perfettini, Jean-Luc

    2011-08-29

    Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.

  4. Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection

    PubMed Central

    Séror, Claire; Melki, Marie-Thérèse; Subra, Frédéric; Raza, Syed Qasim; Bras, Marlène; Saïdi, Héla; Nardacci, Roberta; Voisin, Laurent; Paoletti, Audrey; Law, Frédéric; Martins, Isabelle; Amendola, Alessandra; Abdul-Sater, Ali A.; Ciccosanti, Fabiola; Delelis, Olivier; Niedergang, Florence; Thierry, Sylvain; Said-Sadier, Najwane; Lamaze, Christophe; Métivier, Didier; Estaquier, Jérome; Fimia, Gian Maria; Falasca, Laura; Casetti, Rita; Modjtahedi, Nazanine; Kanellopoulos, Jean; Mouscadet, Jean-François; Ojcius, David M.; Piacentini, Mauro; Gougeon, Marie-Lise

    2011-01-01

    Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches. PMID:21859844

  5. Role of ATP as a Key Signaling Molecule Mediating Radiation-Induced Biological Effects

    PubMed Central

    Ohshima, Yasuhiro; Nakatsukasa, Hiroko; Tsukimoto, Mitsutoshi

    2017-01-01

    Adenosine triphosphate (ATP) serves as a signaling molecule for adaptive responses to a variety of cytotoxic agents and plays an important role in mediating the radiation stress-induced responses that serve to mitigate or repair the injurious effects of γ radiation on the body. Indeed, low doses of radiation may have a net beneficial effect by activating a variety of protective mechanisms, including antitumor immune responses. On the other hand, ATP signaling may be involved in the radiation resistance of cancer cells. Here, focusing on our previous work, we review the evidence that low-dose γ irradiation (0.25-0.5 Gy) induces release of extracellular ATP, and that the released ATP mediates multiple radiation-induced responses, including increased intracellular antioxidant synthesis, cell-mediated immune responses, induction of DNA damage repair systems, and differentiation of regulatory T cells. PMID:28250717

  6. A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

    PubMed

    Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

    2014-08-07

    A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

  7. Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging

    NASA Astrophysics Data System (ADS)

    Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

    2015-04-01

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells. Electronic supplementary information (ESI) available: Supplementary figures related to characterization, computational studies and protein conjugation. See DOI: 10.1039/c5nr01519g

  8. Adenosine effects on inhibitory synaptic transmission and excitation–inhibition balance in the rat neocortex

    PubMed Central

    Zhang, Pei; Bannon, Nicholas M; Ilin, Vladimir; Volgushev, Maxim; Chistiakova, Marina

    2015-01-01

    Abstract Adenosine might be the most widespread neuromodulator in the brain: as a metabolite of ATP it is present in every neuron and glial cell. However, how adenosine affects operation of neurons and networks in the neocortex is poorly understood, mostly because modulation of inhibitory transmission by adenosine has been so little studied. To clarify adenosine's role at inhibitory synapses, and in excitation–inhibition balance in pyramidal neurons, we recorded pharmacologically isolated inhibitory responses, compound excitatory–inhibitory responses and spontaneous events in layer 2/3 pyramidal neurons in slices from rat visual cortex. We show that adenosine (1–150 μm) suppresses inhibitory transmission to these neurons in a concentration-dependent and reversible manner. The suppression was mediated by presynaptic A1 receptors (A1Rs) because it was blocked by a selective A1 antagonist, DPCPX, and associated with changes of release indices: paired-pulse ratio, inverse coefficient of variation and frequency of miniature events. At some synapses (12 out of 24) we found evidence for A2ARs: their blockade led to a small but significant increase of the magnitude of adenosine-mediated suppression. This effect of A2AR blockade was not observed when A1Rs were blocked, suggesting that A2ARs do not have their own effect on transmission, but can modulate the A1R-mediated suppression. At both excitatory and inhibitory synapses, the magnitude of A1R-mediated suppression and A2AR–A1R interaction expressed high variability, suggesting high heterogeneity of synapses in the sensitivity to adenosine. Adenosine could change the balance between excitation and inhibition at a set of inputs to a neuron bidirectionally, towards excitation or towards inhibition. On average, however, these bidirectional changes cancelled each other, and the overall balance of excitation and inhibition was maintained during application of adenosine. These results suggest that changes of adenosine

  9. Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways

    PubMed Central

    Button, Brian; Okada, Seiko F.; Frederick, Charles Brandon; Thelin, William R.; Boucher, Richard C.

    2013-01-01

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal auto-crine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis. PMID:23757023

  10. Mechanosensitive ATP release maintains proper mucus hydration of airways.

    PubMed

    Button, Brian; Okada, Seiko F; Frederick, Charles Brandon; Thelin, William R; Boucher, Richard C

    2013-06-11

    The clearance of mucus from the airways protects the lungs from inhaled noxious and infectious materials. Proper hydration of the mucus layer enables efficient mucus clearance through beating of cilia on airway epithelial cells, and reduced clearance of excessively concentrated mucus occurs in patients with chronic obstructive pulmonary disease and cystic fibrosis. Key steps in the mucus transport process are airway epithelia sensing and responding to changes in mucus hydration. We reported that extracellular adenosine triphosphate (ATP) and adenosine were important luminal autocrine and paracrine signals that regulated the hydration of the surface of human airway epithelial cultures through their action on apical membrane purinoceptors. Mucus hydration in human airway epithelial cultures was sensed by an interaction between cilia and the overlying mucus layer: Changes in mechanical strain, proportional to mucus hydration, regulated ATP release rates, adjusting fluid secretion to optimize mucus layer hydration. This system provided a feedback mechanism by which airways maintained mucus hydration in an optimum range for cilia propulsion. Understanding how airway epithelia can sense and respond to changes in mucus properties helps us to understand how the mucus clearance system protects the airways in health and how it fails in lung diseases such as cystic fibrosis.

  11. Enzyme-responsive polymeric supra-amphiphiles formed by the complexation of chitosan and ATP.

    PubMed

    Kang, Yuetong; Wang, Chao; Liu, Kai; Wang, Zhiqiang; Zhang, Xi

    2012-10-16

    Chitosan and adenosine-5'-triphosphate (ATP) are employed as building blocks to fabricate polymeric supra-amphiphiles based on electrostatic interactions, which can self-assemble to form spherical aggregates. The spherical aggregates inherit the phosphotase responsiveness of ATP. Compared to our previous work, this enzyme-responsive system can be more biocompatible and block polymers are not needed in preparation, which makes it possible to fabricate the chitosan-based enzyme-responsive assemblies in a large-scale, cheap way. Therefore, the application of the assemblies for nanocontainers and drug delivery is greatly anticipated.

  12. Pathological overproduction: the bad side of adenosine.

    PubMed

    Borea, Pier Andrea; Gessi, Stefania; Merighi, Stefania; Vincenzi, Fabrizio; Varani, Katia

    2017-03-02

    Adenosine is an endogenous ubiquitous purine nucleoside, which is increased by hypoxia, ischaemia and tissue damage and mediates a number of physiopathological effects by interacting with four GPCRs, identified as A1 , A2A , A2B and A3 . Physiological and acutely increased adenosine is mostly associated with beneficial effects that include vasodilatation and a decrease in inflammation. In contrast, chronic overproduction of adenosine occurs in important pathological states, where long-lasting increases in the nucleoside levels are responsible for the bad side of adenosine associated with chronic inflammation, fibrosis and organ damage. In this review, we describe and critically discuss the pathological overproduction of adenosine and analyse when, where and how adenosine exerts its detrimental effects throughout the body.

  13. Adenosine triphosphate inhibits melatonin synthesis in the rat pineal gland.

    PubMed

    Souza-Teodoro, Luis Henrique; Dargenio-Garcia, Letícia; Petrilli-Lapa, Camila Lopes; Souza, Ewerton da Silva; Fernandes, Pedro A C M; Markus, Regina P; Ferreira, Zulma S

    2016-03-01

    Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects.

  14. Self-Assembled Tb(3+) Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo.

    PubMed

    Jung, Sung Ho; Kim, Ka Young; Lee, Ji Ha; Moon, Cheol Joo; Han, Noh Soo; Park, Su-Jin; Kang, Dongmin; Song, Jae Kyu; Lee, Shim Sung; Choi, Myong Yong; Jaworski, Justyn; Jung, Jong Hwa

    2017-01-11

    To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C3 symmetrical terpyridine complex with Tb(3+) (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H2PO4(-) (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated.

  15. Adenosine triphosphate acts as a paracrine signaling molecule to reduce the motility of T cells.

    PubMed

    Wang, Chiuhui Mary; Ploia, Cristina; Anselmi, Fabio; Sarukhan, Adelaida; Viola, Antonella

    2014-06-17

    Organization of immune responses requires exchange of information between cells. This is achieved through either direct cell-cell contacts and establishment of temporary synapses or the release of soluble factors, such as cytokines and chemokines. Here we show a novel form of cell-to-cell communication based on adenosine triphosphate (ATP). ATP released by stimulated T cells induces P2X4/P2X7-mediated calcium waves in the neighboring lymphocytes. Our data obtained in lymph node slices suggest that, during T-cell priming, ATP acts as a paracrine messenger to reduce the motility of lymphocytes and that this may be relevant to allow optimal tissue scanning by T cells.

  16. Optogenetic control of ATP release

    NASA Astrophysics Data System (ADS)

    Lewis, Matthew A.; Joshi, Bipin; Gu, Ling; Feranchak, Andrew; Mohanty, Samarendra K.

    2013-03-01

    Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells, these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly, photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic signaling.

  17. Effect of adenosine 5'-triphosphate infusions on the nutritional status and survival of preterminal cancer patients.

    PubMed

    Beijer, Sandra; Hupperets, Pierre S; van den Borne, Ben E; Eussen, Simone R; van Henten, Arjen M; van den Beuken-van Everdingen, Marieke; de Graeff, Alexander; Ambergen, Ton A; van den Brandt, Piet A; Dagnelie, Pieter C

    2009-08-01

    The aim of the study was to investigate the effect of intravenous infusions of adenosine 5'-triphosphate (ATP) on nutritional status and survival in preterminal cancer patients. Ninety-nine preterminal cancer patients (estimated life expectancy 1-6 months) with mixed tumor types were randomly allocated to receive either intravenous ATP weekly (8-10 h/week, maximum 50 microg/kg/min) for 8 weeks, or no ATP (control group). Nutritional status parameters were assessed until 8 weeks, and analyzed by repeated-measures analysis of covariance. Cox proportional hazards models were fitted to assess the effect of ATP on short-term (0-8 weeks) and long-term (0-6 months) survival. Fifty-one patients were randomized to ATP and 48 to the control group. Results showed a significant favorable effect of ATP on triceps skin fold thickness [between-group difference per 8 weeks 1.76 mm, 95% confidence interval (CI): 0.48-3.12 mm; P = 0.009] and on short-term survival [0-8 weeks hazard ratio (HR): 0.40, 95% CI: 0.17-0.95; P = 0.037]. In weight-stable patients and in lung cancer patients, long-term survival (0-6 months) was also significantly better in ATP-treated patients (weight-stable patients HR: 0.40, 95% CI: 0.19-0.83; P = 0.014; patients with lung cancer: HR: 0.35, 95% CI: 0.14-0.88; P = 0.025). In conclusion, in this population of preterminal cancer patients, ATP infusions, at the dose and schedule studied, had a favorable effect on triceps skin fold thickness and survival, especially in weight-stable patients and patients with lung cancer. Larger studies are warranted to confirm these findings and to further define the effect of ATP on tumor growth and survival.

  18. Torsades de pointes after adenosine administration.

    PubMed

    Teodorovich, Nicholay; Margolin, Elena; Kogan, Yonatan; Paz, Ofir; Swissa, Moshe

    2016-01-01

    Adenosine can produce arrhythmias, which are generally short living. It may induce PACs and PVCs, sinus bradycardia, and atrial fibrillation. There have been reports of transient polymorphic VT (torsades de pointes) in patients with LQTS and others in people with normal QT interval. We report a case of a long episode of polymorphic VT induced by adenosine. A 27 year old woman received 6 mg adenosine for PSVT, which terminated and torsades de pointes developed, persisting for 17 seconds and terminated spontaneously. This is the longest described duration of the torsades after adenosine administration in patients with normal QT interval.

  19. Galactosylated chitosan oligosaccharide nanoparticles for hepatocellular carcinoma cell-targeted delivery of adenosine triphosphate.

    PubMed

    Zhu, Xiu Liang; Du, Yong Zhong; Yu, Ri Sheng; Liu, Ping; Shi, Dan; Chen, Ying; Wang, Ying; Huang, Fang Fang

    2013-07-29

    Nanoparticles composed of galactosylated chitosan oligosaccharide (Gal-CSO) and adenosine triphosphate (ATP) were prepared for hepatocellular carcinoma cell-specific uptake, and the characteristics of Gal-CSO/ATP nanoparticles were evaluated. CSO/ATP nanoparticles were prepared as a control. The average diameter and zeta potential of Gal-CSO/ATP nanoparticles were 51.03 ± 3.26 nm and 30.50 ± 1.25 mV, respectively, suggesting suitable properties for a drug delivery system. Subsequently, the cytotoxicity of Gal-CSO/ATP nanoparticles were examined by the methyl tetrazolium (MTT) assay, and the half maximal inhibitory concentration (IC50) values were calculated with HepG2 (human hepatocellular carcinoma cell line) cells. The results showed that the cytotoxic effect of nanoparticles on HepG2 cells was low. In the meantime, it was also found that the Gal-CSO/ATP nanoparticles could be uptaken by HepG2 cells, due to expression of the asialoglycoprotein receptor (ASGP-R) on their surfaces. The presented results indicate that the Gal-CSO nanoparticles might be very attractive to be used as an intracellular drug delivery carrier for hepatocellular carcinoma cell targeting, thus warranting further in vivo or clinical investigations.

  20. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

    PubMed

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

    2015-01-01

    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  1. Improving environmental cleaning in clinical areas: staff education based on adenosine triphosphate readings.

    PubMed

    Villanueva, Ariadna; Guanche, Humberto

    2016-11-01

    Aim To describe the effect of education on environmental cleaning in patient care areas using adenosine triphosphate (ATP) readings. Method A quality improvement initiative was developed in a community hospital in Qatar. Over a two-month period, an infection-control practitioner monitored ATP readings in patient care areas, at any time and regardless of the time of the previous disinfection. The initiative included staff education, use of ATP readings and the drawing up of quarterly quality reports. The ATP readings were considered 'pass', meaning well cleaned, or 'fail', meaning non-cleaned, according to the following standards:>250 relative light units (RLU) in non-critical units and<200RLU for critical units. The proportion of test passes was calculated per 100 tests performed. Results A total of 1,617 tests were performed, after which 1,259 (78%) surfaces were identified as well cleaned. The lowest proportion of non-pass and higher ATP readings was observed in non-critical areas. The test points with the lowest proportion of passes were telephones (40.5%), a medication dispensing system (58.5%), an oximeter (66.7%) and callbox buttons (67.6%). A sustained increase in test passes was observed during the study period. Conclusion There was an improvement in environmental cleaning due to monitoring of ATP on surfaces and staff education.

  2. Electrochemiluminescence aptasensor for adenosine triphosphate detection using host-guest recognition between metallocyclodextrin complex and aptamer.

    PubMed

    Chen, Hong; Chen, Qiong; Zhao, Yingying; Zhang, Fan; Yang, Fan; Tang, Jie; He, Pingang

    2014-04-01

    A sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed using host-guest recognition between a metallocyclodextrin complex, i.e., tris(bipyridine)ruthenium(II)-β-cyclodextrin [tris(bpyRu)-β-CD], and an ATP-binding aptamer. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition between tris(bpyRu)-β-CD and aptamer, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration was obtained in the range 10.0-0.05 nM, with a detection limit of 0.01 nM at the S/N ratio of 3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets.

  3. Double-functionalized gold nanoparticles with split aptamer for the detection of adenosine triphosphate.

    PubMed

    Cheng, Sheng; Zheng, Bin; Wang, Mozhen; Lam, Michael Hon-Wah; Ge, Xuewu

    2013-10-15

    A newly designed functionalization type for gold nanoparticles (AuNP) with split aptamer has been developed for the detection of adenosine triphosphate (ATP). The ATP aptamer was split into two parts with their 5' prime or 3' prime modified with thiol. Both the 5' SH and 3' SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA conjugates. Thus, the split aptamer can be reassembled into intact folded structure in the presence of ATP molecule with two potential assembly types, which induces the assembly of AuNP-DNA conjugates. In this double-functionalized system, the traditional assembly type might facilitate another assembly type, which was found to give much higher LSPR change in the presence of ATP than the traditional assembly type, and improve the sensitivity for ATP detection. Time courses of the assemble processes with different assembly types, Mg(2+) concentrations, and aptamer fragments densities on AuNP were followed using the absorption ratio at 650 nm and 520 nm. ATP response with this newly designed system was investigated using absorption spectra and dynamic light scattering method.

  4. Light scattering change precedes loss of cerebral adenosine triphosphate in a rat global ischemic brain model.

    PubMed

    Kawauchi, Satoko; Sato, Shunichi; Ooigawa, Hidetoshi; Nawashiro, Hiroshi; Ishihara, Miya; Kikuchi, Makoto

    2009-08-14

    Measurement of intrinsic optical signals (IOSs) is an attractive technique for monitoring tissue viability in brains since it enables noninvasive, real-time monitoring of morphological characteristics as well as physiological and biochemical characteristics of tissue. We previously showed that light scattering signals reflecting cellular morphological characteristics were closely related to the IOSs associated with the redox states of cytochrome c oxidase in the mitochondrial respiratory chain. In the present study, we examined the relationship between light scattering and energy metabolism. Light scattering signals were transcranially measured in rat brains after oxygen and glucose deprivation, and the results were compared with concentrations of cerebral adenosine triphosphate (ATP) measured by luciferin-luciferase bioluminescence assay. Electrophysiological signal was also recorded simultaneously. After starting saline infusion, EEG activity ceased at 108+/-17s, even after which both the light scattering signal and ATP concentration remained at initial levels. However, light scattering started to change in three phases at 236+/-15s and then cerebral ATP concentration started to decrease at about 260s. ATP concentration significantly decreased during the triphasic scattering change, indicating that the start of scattering change preceded the loss of cerebral ATP. The mean time difference between the start of triphasic scattering change and the onset of ATP loss was about 24s in the present model. DC potential measurement showed that the triphasic scattering change was associated with anoxic depolarization. These findings suggest that light scattering signal can be used as an indicator of loss of tissue viability in brains.

  5. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  6. Insulin modulation of ATP-sensitive K+ channel of rat skeletal muscle is impaired in the hypokalaemic state.

    PubMed

    Tricarico, D; Capriulo, R; Conte Camerino, D

    1999-01-01

    In the present work, we examined the effects of in vivo administration of insulin to rats made hypokalaemic by feeding a K+-free diet. The i.p. injection of insulin in the hypokalaemic rats provoked muscle paralysis within 3-5 h. Consistent with this observation, the skeletal muscle fibres of the paralysed rats were depolarized. In contrast, in the normokalaemic animals, insulin neither provoked paralysis nor produced significant fibre hyperpolarization. In the hypokalaemic rats, insulin almost completely abolished the sarcolemma adenosine triphosphate (ATP)-sensitive K+ currents without altering the sensitivity of the channels to ATP or glibenclamide. In contrast, in the normokalaemic rats, insulin enhanced ATP-sensitive K+ currents that became also resistant to ATP and glibenclamide. Our experiments indicate that the modulation of the sarcolemma ATP-sensitive K+ channels by insulin is impaired in the hypokalaemic state. This phenomenon appears to be related to the fibre depolarization and paralysis observed in the same animals.

  7. Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

    PubMed

    Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Ray, Sougata Sinha; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

    2015-03-01

    Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

  8. Different danger signals differently impact on microglial proliferation through alterations of ATP release and extracellular metabolism.

    PubMed

    George, Jimmy; Gonçalves, Francisco Q; Cristóvão, Gonçalo; Rodrigues, Lisa; Meyer Fernandes, José Roberto; Gonçalves, Teresa; Cunha, Rodrigo A; Gomes, Catarina A

    2015-09-01

    Microglia rely on their ability to proliferate in the brain parenchyma to sustain brain innate immunity and participate in the reaction to brain damage. We now studied the influence of different danger signals activating microglia, both internal (typified by glutamate, associated with brain damage) and external (using a bacterial lipopolysaccharide, LPS), on the proliferation of microglia cells. We found that LPS (100 ng/mL) increased, whereas glutamate (0.5 mM) decreased proliferation. Notably, LPS decreased whereas glutamate increased the extracellular levels of ATP. In contrast, LPS increased whereas glutamate decreased the extracellular catabolism of ATP into adenosine through ecto-nucleotidases and ecto-5'-nucleotidase. Finally, apyrase (degrades extracellular ATP) abrogated glutamate-induced inhibition of microglia proliferation; conversely, inhibitors of ecto-nucleotidases (ARL67156 or α,β-methylene ADP) and adenosine deaminase (degrades extracellular adenosine) abrogated the LPS-induced increase of microglia proliferation, which was blocked by a selective A2A receptor antagonist, SCH58261 (50 nM). Overall, these results highlight the importance of the extracellular purinergic metabolism to format microglia proliferation and influence the spatio-temporal profile of neuroinflammation in different conditions of brain damage.

  9. Mechanical effects of muscle contraction increase intravascular ATP draining quiescent and active skeletal muscle in humans.

    PubMed

    Crecelius, Anne R; Kirby, Brett S; Richards, Jennifer C; Dinenno, Frank A

    2013-04-01

    Intravascular adenosine triphosphate (ATP) evokes vasodilation and is implicated in the regulation of skeletal muscle blood flow during exercise. Mechanical stresses to erythrocytes and endothelial cells stimulate ATP release in vitro. How mechanical effects of muscle contractions contribute to increased plasma ATP during exercise is largely unexplored. We tested the hypothesis that simulated mechanical effects of muscle contractions increase [ATP](venous) and ATP effluent in vivo, independent of changes in tissue metabolic demand, and further increase plasma ATP when superimposed with mild-intensity exercise. In young healthy adults, we measured forearm blood flow (FBF) (Doppler ultrasound) and plasma [ATP](v) (luciferin-luciferase assay), then calculated forearm ATP effluent (FBF×[ATP](v)) during rhythmic forearm compressions (RFC) via a blood pressure cuff at three graded pressures (50, 100, and 200 mmHg; Protocol 1; n = 10) and during RFC at 100 mmHg, 5% maximal voluntary contraction rhythmic handgrip exercise (RHG), and combined RFC + RHG (Protocol 2; n = 10). [ATP](v) increased from rest with each cuff pressure (range 144-161 vs. 64 ± 13 nmol/l), and ATP effluent was graded with pressure. In Protocol 2, [ATP](v) increased in each condition compared with rest (RFC: 123 ± 33; RHG: 51 ± 9; RFC + RHG: 96 ± 23 vs. Mean Rest: 42 ± 4 nmol/l; P < 0.05), and ATP effluent was greatest with RFC + RHG (RFC: 5.3 ± 1.4; RHG: 5.3 ± 1.1; RFC + RHG: 11.6 ± 2.7 vs. Mean Rest: 1.2 ± 0.1 nmol/min; P < 0.05). We conclude that the mechanical effects of muscle contraction can 1) independently elevate intravascular ATP draining quiescent skeletal muscle without changes in local metabolism and 2) further augment intravascular ATP during mild exercise associated with increases in metabolism and local deoxygenation; therefore, it is likely one stimulus for increasing intravascular ATP during exercise in humans.

  10. Natural Variation in the ATPS1 Isoform of ATP Sulfurylase Contributes to the Control of Sulfate Levels in Arabidopsis1[W

    PubMed Central

    Koprivova, Anna; Giovannetti, Marco; Baraniecka, Patrycja; Lee, Bok-Rye; Grondin, Cécile; Loudet, Olivier; Kopriva, Stanislav

    2013-01-01

    Sulfur is an essential macronutrient for all living organisms. Plants take up inorganic sulfate from the soil, reduce it, and assimilate it into bioorganic compounds, but part of this sulfate is stored in the vacuoles. In our first attempt to identify genes involved in the control of sulfate content in the leaves, we reported that a quantitative trait locus (QTL) for sulfate content in Arabidopsis (Arabidopsis thaliana) was underlain by the APR2 isoform of the key enzyme of sulfate assimilation, adenosine 5′-phosphosulfate reductase. To increase the knowledge of the control of this trait, we cloned a second QTL from the same analysis. Surprisingly, the gene underlying this QTL encodes the ATPS1 isoform of the enzyme ATP sulfurylase, which precedes adenosine 5′-phosphosulfate reductase in the sulfate assimilation pathway. Plants with the Bay allele of ATPS1 accumulate lower steady-state levels of ATPS1 transcript than those with the Sha allele, which leads to lower enzyme activity and, ultimately, the accumulation of sulfate. Our results show that the transcript variation is controlled in cis. Examination of ATPS1 sequences of Bay-0 and Shahdara identified two deletions in the first intron and immediately downstream the gene in Bay-0 shared with multiple other Arabidopsis accessions. The average ATPS1 transcript levels are lower in these accessions than in those without the deletions, while sulfate levels are significantly higher. Thus, sulfate content in Arabidopsis is controlled by two genes encoding subsequent enzymes in the sulfate assimilation pathway but using different mechanisms, variation in amino acid sequence and variation in expression levels. PMID:24027241

  11. Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities

    PubMed Central

    Anderson, Catherine B.; Kinnamon, Sue C.

    2015-01-01

    Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. PMID:26136251

  12. The reaction process of firefly bioluminescence triggered by photolysis of caged-ATP.

    PubMed

    Kageyama, Takeshi; Tanaka, Masatoshi; Sekiya, Takao; Ohno, Shin-Ya; Wada, Naohisa

    2011-01-01

    The reaction process of firefly bioluminescence was studied by photolyzing caged-ATP to adenosine triphosphate (ATP) within 100 ms. The intensity of luminescence increases markedly to reach a maximum within 1 s, maintains almost the same intensity up to 5 s and then decays monotonically. The rise γ(1) and decay γ(2) rate constants were determined to be about 5 s(-1) and 1 × 10(-2) s(-1), respectively, so as to phenomenologically fit the time course. A second luminescence peak appears after around 350 s. The dependence of the rate constants on the concentrations of reactants and a viscous reagent revealed that two kinds of reaction contribute the observed time course: (1) an intrinsic reaction by ATP photolyzed from caged-ATP that is already trapped in luciferase; and (2) a diffusion-controlled reaction by free ATP in the buffer solution outside luciferase. Numerical analysis based on reaction kinetics related γ(1) and γ(2) to the rate constants of a three-step reaction model, and accurately described the effects of concentration of reactants and a viscous reagent on the time courses of bioluminescence. Thus, it has been clearly concluded that the binding mode of caged-ATP at the catalytic center of luciferase is very different from that of ATP.

  13. Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

    PubMed

    Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C

    2015-09-01

    Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli.

  14. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

    PubMed Central

    Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

    2015-01-01

    Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

  15. ATP monitoring technology for microbial growth control in potable water systems

    NASA Astrophysics Data System (ADS)

    Whalen, Patrick A.; Whalen, Philip J.; Cairns, James E.

    2006-05-01

    ATP (Adenosine Triphosphate) is the primary energy transfer molecule present in all living biological cells on Earth. ATP cannot be produced or maintained by anything but a living organism, and as such, its measurement is a direct indication of biological activity. The main advantage of ATP as a biological indicator is the speed of the analysis - from collecting the sample to obtaining the result, only minutes are required. The technology to measure ATP is already widely utilized to verify disinfection efficacy in the food industry and is also commonly applied in industrial water processes such as cooling water systems to monitor microbial growth and biocide applications. Research has indicated that ATP measurement technology can also play a key role in such important industries as potable water distribution and biological wastewater treatment. As will be detailed in this paper, LuminUltra Technologies has developed and applied ATP measurement technologies designed for any water type, and as such can provide a method to rapidly and accurately determine the level of biological activity in drinking water supplies. Because of its speed and specificity to biological activity, ATP measurement can play a key role in defending against failing drinking water quality, including those encountered during routine operation and also bioterrorism.

  16. Adenosine Triphosphate-Triggered Release of Macromolecular and Nanoparticle Loads from Aptamer/DNA-Cross-Linked Microcapsules.

    PubMed

    Liao, Wei-Ching; Lu, Chun-Hua; Hartmann, Raimo; Wang, Fuan; Sohn, Yang Sung; Parak, Wolfgang J; Willner, Itamar

    2015-09-22

    The synthesis of stimuli-responsive DNA microcapsules acting as carriers for different payloads, and being dissociated through the formation of aptamer-ligand complexes is described. Specifically, stimuli-responsive anti-adenosine triphosphate (ATP) aptamer-cross-linked DNA-stabilized microcapsules loaded with tetramethylrhodamine-modified dextran (TMR-D), CdSe/ZnS quantum dots (QDs), or microperoxidase-11 (MP-11) are presented. In the presence of ATP as trigger, the microcapsules are dissociated through the formation of aptamer-ATP complexes, resulting in the release of the respective loads. Selective unlocking of the capsules is demonstrated, and CTP, GTP, or TTP do not unlock the pores. The ATP-triggered release of MP-11 from the microcapsules enables the MP-11-catalyzed oxidation of Amplex UltraRed by H2O2 to the fluorescent product resorufin.

  17. Adenosine monophosphate-activated protein kinase: a central regulator of metabolism with roles in diabetes, cancer, and viral infection.

    PubMed

    Hardie, D G

    2011-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor activated by metabolic stresses that inhibit catabolic ATP production or accelerate ATP consumption. Once activated, AMPK switches on catabolic pathways, generating ATP, while inhibiting cell growth and proliferation, thus promoting energy homeostasis. AMPK is activated by the antidiabetic drug metformin, and by many natural products including "nutraceuticals" and compounds used in traditional medicines. Most of these xenobiotics activate AMPK by inhibiting mitochondrial ATP production. AMPK activation by metabolic stress requires the upstream kinase, LKB1, whose tumor suppressor effects may be largely mediated by AMPK. However, many tumor cells appear to have developed mechanisms to reduce AMPK activation and thus escape its growth-restraining effects. A similar phenomenon occurs during viral infection. If we can establish how down-regulation occurs in tumors and virus-infected cells, there may be therapeutic avenues to reverse these effects.

  18. Activated sludge optimization using ATP in pulp and paper industry.

    PubMed

    Bäckman, Göran; Gytel, Ulla

    2015-01-01

    The activated sludge process is an old technology, but still the most commonly used one for treatment of wastewater. Despite the wide spread usage the technology still suffers from instability (Tandoi et al. 2006) and high operating cost. Activated sludge processes often carry a large solids inventory. Managing the total inventory without interference is the key component of the optimization process described in this paper. Use of nutrients is common in pulp and paper effluent treatment. Feeding enough nutrients to support the biomass growth is a delicate balance. Overfeeding or underfeeding of nutrients can result in higher costs. Detrimental substances and toxic components in effluents entering a biological treatment system can cause severe, long lasting disturbances (Hynninen & Ingman 1998; Bergeron & Pelletier 2004). A LumiKem test kit is used to measure biological activity with adenosine triphosphate (ATP) in a pulp and paper mill. ATP data are integrated with other standardized mill parameters. Measurements of active volatile suspended solids based on ATP can be used to quantify the living biomass in the activated sludge process and to ensure that sufficient biomass is present in order to degrade the wastewater constituents entering the process. Information about active biomass will assist in optimizing sludge inventories and feeding of nutrients allowing the living biomass to re-populate to create optimal efficiency. ATP measurements can also be used to alert operators if any components toxic to bacteria are present in wastewater. The bio stress index represents the stress level experienced by the microbiological population. This parameter is very useful in monitoring toxicity in and around bioreactors. Results from the wastewater process optimization and ATP measurements showed that treatment cost could be reduced by approximately 20-30% with fewer disturbances and sustained biological activity compared to the reference period. This was mainly achieved by

  19. Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors.

    PubMed

    Panjehpour, Mojtaba; Karami-Tehrani, Fatemeh

    2007-01-01

    Adenosine modulates the proliferation, survival, and apoptosis of many different cell types. The present study was performed to investigate the role of adenosine receptors in the human breast cancer cell lines MCF-7 and MDA-MB468. The biological effects of adenosine on the cells were analyzed by adenylyl cyclase and cell viability assay as well as RT-PCR of adenosine receptors. RT-PCR results show the expression of the transcript of all adenosine receptors in both cell lines. By using adenosine and selective adenosine receptor agonists or antagonists, we found that A3 stimulation reduced cell viability, which was abolished by pretreatment with A3 receptor antagonist. Moreover, we demonstrated that adenosine (natural agonist) triggers a cytotoxic signal via A3 receptor activation that was not seen for other subclasses of adenosine receptors. Intracellular cAMP concentration was changed significantly only for A3 and A2B receptor-selective agonists, which indicates the functional form of these receptors on the cell surface. In conclusion, our findings revealed the role of adenosine receptors in breast cancer cell lines on growth modulation role of A3 and functional form of A2B, although its involvement in cell growth modulation was not seen. Theses findings as well as data by others may provide a possible application of adenosine receptor agonists/antagonists in breast malignancies.

  20. Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina

    SciTech Connect

    Braas, K.M.; Zarbin, M.A.; Snyder, S.H.

    1987-06-01

    Using specific sensitive antisera against adenosine, we have immunocytochemically localized endogenous adenosine to specific layers of rat, guinea pig, monkey, and human retina. Highest adenosine immunoreactivity was observed in ganglion cells and their processes in the optic nerve fiber layer. Substantial staining was also found throughout the inner plexiform layer and in select cells in the inner nuclear layer. Adenosine A1 receptors, labeled with the agonists L-(/sup 3/H)phenylisopropyladenosine and /sup 125/I-labeled hydroxy-phenylisopropyladenosine, were autoradiographically localized. The highest levels of binding sites occurred in the nerve fiber, ganglion cell, and inner plexiform layers of the retina in all the species examined. The distribution of adenosine A1 receptor sites closely parallels that of retinal neurons and fibers containing immunoreactive adenosine. These results suggest a role for endogenous adenosine as a coneurotransmitter in ganglion cells and their fibers in the optic nerve.

  1. Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis.

    PubMed Central

    Shrimankar, P; Stordal, L; Maurer, R

    1992-01-01

    The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme. Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase. While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment. To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins. Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231. The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB. Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action. It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication. In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers. On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination. Images PMID:1332941

  2. Novel Antibiotic Susceptibility Tests by the ATP-Bioluminescence Method Using Filamentous Cell Treatment

    PubMed Central

    Hattori, Noriaki; Nakajima, Moto-O; O’Hara, Koji; Sawai, Tetsuo

    1998-01-01

    Antimicrobial susceptibility testing by the ATP-bioluminescence method has been noted for its speed; it provides susceptibility results within 2 to 5 h. However, several disagreements between the ATP method and standard methodology have been reported. The present paper describes a novel ATP method in a 3.5-h test which overcomes these deficiencies through the elimination of false-resistance discrepancies in tests on gram-negative bacteria with β-lactam agents. In our test model using Pseudomonas aeruginosa and piperacillin, it was shown that ATP in filamentous cells accounted for the false resistance. We found that 0.5% 2-amino-2-methyl-1,3-propanediol (AMPD) extracted ATP from the filamentous cells without affecting normal cells and that 0.3 U of adenosine phosphate deaminase (APDase)/ml simultaneously digested the extracted ATP. We used the mixture of these reagents for the pretreatment of cells in a procedure we named filamentous cell treatment, prior to ATP measurements. This novel ATP method with the filamentous cell treatment eliminated false-resistance discrepancies in tests on P. aeruginosa with β-lactam agents, including piperacillin, cefoperazone, aztreonam, imipenem-cilastatin, ceftazidime, and cefsulodin. Furthermore, this novel methodology produced results which agreed with those of the standard microdilution method in other tests on gram-negative and gram-positive bacteria, including P. aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, for non-β-lactam agents, such as fosfomycin, ofloxacin, minocycline, and aminoglycosides. MICs obtained by the novel ATP method were also in agreement with those obtained by the agar dilution method of susceptibility testing. From these results, it was shown that the novel ATP method could be used successfully to test the activities of antimicrobial agents with the elimination of the previously reported discrepancies. PMID:9624485

  3. ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation.

    PubMed

    Verrecchia, F; Duthe, F; Duval, S; Duchatelle, I; Sarrouilhe, D; Herve, J C

    1999-04-15

    1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

  4. Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide.

    PubMed

    Caiazzo, Elisabetta; Maione, Francesco; Morello, Silvana; Lapucci, Andrea; Paccosi, Sara; Steckel, Bodo; Lavecchia, Antonio; Parenti, Astrid; Iuvone, Teresa; Schrader, Jürgen; Ialenti, Armando; Cicala, Carla

    2016-07-15

    Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73

  5. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    SciTech Connect

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  6. Adenosine 5' triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve.

    PubMed

    James, G; Butt, A M

    1999-06-11

    Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5' triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 microm ATP, and with a rank order of agonist potency of ATP > ADP > alpha,beta-methyline-ATP > UDP > adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

  7. Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium.

    PubMed

    Yuan, Yang; Peng, Li; Gong-Hua, Hu; Lu, Dai; Xia-Li, Zhong; Yu, Zhou; Cai-Gao, Zhong

    2012-06-01

    This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

  8. Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors.

    PubMed

    Shinozaki, Youichi; Koizumi, Schuichi; Ishida, Seiichi; Sawada, Jun-Ichi; Ohno, Yasuo; Inoue, Kazuhide

    2005-01-15

    Oxidative stress is the main cause of neuronal damage in traumatic brain injury, hypoxia/reperfusion injury, and neurodegenerative disorders. Although extracellular nucleosides, especially adenosine, are well known to protect against neuronal damage in such pathological conditions, the effects of these nucleosides or nucleotides on glial cell damage remain largely unknown. We report that ATP but not adenosine protects against the cell death of cultured astrocytes induced by hydrogen peroxide (H2O2). ATP ameliorated the H2O2-induced decrease in cell viability of astrocytes in an incubation time- and concentration-dependent fashion. Protection by ATP was inhibited by P2 receptor antagonists and was mimicked by P2Y1 receptor agonists but not by adenosine. The expressions of P2Y1 mRNAs and functional P2Y1 receptors in astrocytes were confirmed. Thus, ATP, acting on P2Y1 receptors in astrocytes, showed a protective action against H2O2. The astrocytic protection by the P2Y1 receptor agonist 2-methylthio-ADP was inhibited by an intracellular Ca2+ chelator and a blocker of phospholipase C, indicating the involvement of intracellular signals mediated by Gq/11-coupled P2Y1 receptors. The ATP-induced protection was inhibited by cycloheximide, a protein synthesis inhibitor, and it took more than 12 h for the onset of the protective action. In the DNA microarray analysis, ATP induced a dramatic upregulation of various oxidoreductase genes. Taken together, ATP acts on P2Y1 receptors coupled to Gq/11, resulting in the upregulation of oxidoreductase genes, leading to the protection of astrocytes against H2O2.

  9. Inhibition of phosphorylation of cardiac troponin-I by adenosine and 5'-chloro 5'-deoxyadenosine

    SciTech Connect

    Rosenthal, R.; Lowenstein, J.M.

    1986-05-01

    Adenosine inhibits the positive inotropic and chronotropic effects of catecholamines on the heart. Incubation of a crude contractile protein fraction from rat heart with (..gamma..-/sup 32/P)ATP results in the phosphorylation of several proteins by endogenous protein kinases. The adenosine analog 5'-chloro 5'-deoxyadenosine inhibits the phosphorylation of a 29 kDa protein in this preparation. The protein has been identified as cardiac troponin-I (cTn-I) by 2 dimensional gel electrophoresis, with a non-equilibrium pH gradient in the first direction and SDS-PAGE in the second, using purified cTn-I as standard. Addition of catalytic subunit of cAMP-dependent protein kinase to the crude contractile fraction increases phosphorylation of cTn-I; this increase is inhibited by 5'-chloro 5'-deoxyadenosine and adenosine. Phosphorylation of purified cTn-I by the catalytic subunit is also inhibited by 5'-chloro 5'-deoxyadenosine. Fifty percent inhibition of phosphorylation by either endogenous or exogenous kinase was observed at approximately 50 ..mu..M 5'-chloro 5'-deoxyadenosine or adenosine, a concentration within the intracellular range during hypoxia.

  10. Adenosine as a Multi-Signalling Guardian Angel in Human Diseases: When, Where and How Does it Exert its Protective Effects?

    PubMed

    Borea, Pier Andrea; Gessi, Stefania; Merighi, Stefania; Varani, Katia

    2016-06-01

    The importance of adenosine for human health cannot be overstated. Indeed, this ubiquitous nucleoside is an integral component of ATP, and regulates the function of every tissue and organ in the body. Acting via receptor-dependent and -independent mechanisms [the former mediated via four G-protein-coupled receptors (GPCRs), A1, A2A, A2B, and A3,], it has a significant role in protecting against cell damage in areas of increased tissue metabolism, and combating organ dysfunction in numerous pathological states. Accordingly, raised levels of adenosine have been demonstrated in epilepsy, ischaemia, pain, inflammation, and cancer, in which its behaviour can be likened to that of a guardian angel, even though there are instances in which overproduction of adenosine is pathological. In this review, we condense the current body of knowledge on the issue, highlighting when, where, and how adenosine exerts its protective effects in both the brain and the periphery.

  11. Lack of correlation between Legionella colonization and microbial population quantification using heterotrophic plate count and adenosine triphosphate bioluminescence measurement.

    PubMed

    Duda, Scott; Baron, Julianne L; Wagener, Marilyn M; Vidic, Radisav D; Stout, Janet E

    2015-07-01

    This investigation compared biological quantification of potable and non-potable (cooling) water samples using pour plate heterotrophic plate count (HPC) methods and adenosine triphosphate (ATP) concentration measurement using bioluminescence. The relationship between these measurements and the presence of Legionella spp. was also examined. HPC for potable and non-potable water were cultured on R2A and PCA, respectively. Results indicated a strong correlation between HPC and ATP measurements in potable water (R = 0.90, p < 0.001). In the make-up water and two cooling towers, the correlations between ATP and HPC were much weaker but statistically significant (make-up water: R = 0.37, p = 0.005; cooling tower 1: R = 0.52, p < 0.001; cooling tower 2: R = 0.54, p < 0.001). For potable and non-potable samples, HPC exhibited higher measurement variability than ATP. However, ATP measurements showed higher microbial concentrations than HPC measurements. Following chlorination of the cooling towers, ATP measurements indicated very low bacterial concentrations (<10 colony-forming units (CFU)/mL) despite high HPC concentrations (>1000 CFU/mL) which consisted primarily of non-tuberculous mycobacteria. HPC concentrations have been suggested to be predictive of Legionella presence, although this has not been proven. Our evaluation showed that HPC or ATP demonstrated a fair predictive capacity for Legionella positivity in potable water (HPC: receiver operating characteristic (ROC) = 0.70; ATP: ROC = 0.78; p = 0.003). However, HPC or ATP correctly classified sites as positive only 64 and 62% of the time, respectively. No correlation between HPC or ATP and Legionella colonization in non-potable water samples was found (HPC: ROC = 0.28; ATP: ROC = 0.44; p = 0.193).

  12. A new strategy for the detection of adenosine triphosphate by aptamer/quantum dot biosensor based on chemiluminescence resonance energy transfer.

    PubMed

    Zhou, Zi-Ming; Yu, Yong; Zhao, Yuan-Di

    2012-09-21

    We designed an aptasensor for the detection of adenosine triphosphate (ATP) based on chemiluminescence resonance energy transfer (CRET). An adenosine aptamer was cut into two pieces of ssDNA, which were attached to quantum dots (QDs) and horse radish peroxidase (HRP), respectively. They could reassemble into specific structures in the presence of ATP and then decrease the distance of HRP and QDs. ATP detection can be easily realized according to the fluorescent intensity of QDs, which is excited by CRET between luminol and QDs. Results show that the concentration of ATP is linear relation with the fluorescent intensity of the peak of QDs emission and the linear range for the linear equation is from 50 μM to 231 μM and the detection limit was 185 nM. When the concentration of ATP was 2 mM, the efficiency of CRET is 13.6%. Good specificity for ATP had been demonstrated compared to thymidine triphosphate (TTP), cytidine triphosphate (CTP) and guanosine triphosphate (GTP), when 1 mM of each was added, respectively. This method needs no external light source and can avoid autofluorescence and photobleaching, and ATP can be detected selectively, specifically, and sensitively in a low micromolar range, which means that the strategy reported here can be applicable to the detection of several other target molecules.

  13. The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

    PubMed Central

    Ferenczi, M A; Homsher, E; Trentham, D R

    1984-01-01

    The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19 degrees C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments. At full overlap and in the absence of calcium (less than 10(-8) M) and nucleotide, the fibres developed rigor tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s-1. In the presence of 2 X 10(-5) M-free calcium ions, the initial phase following the laser flash lasted approximately 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibres at full myofilament overlap, and equal to 0.7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1.8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the

  14. Reexamination of magnetic isotope and field effects on adenosine triphosphate production by creatine kinase

    PubMed Central

    Crotty, Darragh; Silkstone, Gary; Poddar, Soumya; Ranson, Richard; Prina-Mello, Adriele; Wilson, Michael T.; Coey, J. M. D.

    2012-01-01

    The influence of isotopically enriched magnesium on the creatine kinase catalyzed phosphorylation of adenosine diphosphate is examined in two independent series of experiments where adenosine triphosphate (ATP) concentrations were determined by a luciferase-linked luminescence end-point assay or a real-time spectrophotometric assay. No increase was observed between the rates of ATP production with natural Mg, 24Mg, and 25Mg, nor was any significant magnetic field effect observed in magnetic fields from 3 to 1,000 mT. Our results are in conflict with those reported by Buchachenko et al. [J Am Chem Soc 130:12868–12869 (2008)], and they challenge these authors’ general claims that a large (two- to threefold) magnetic isotope effect is “universally observable” for ATP-producing enzymes [Her Russ Acad Sci 80:22–28 (2010)] and that “enzymatic phosphorylation is an ion-radical, electron-spin-selective process” [Proc Natl Acad Sci USA 101:10793–10796 (2005)]. PMID:22198842

  15. Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: a Source of Adenosine Production.

    PubMed

    Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

    2017-01-24

    Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic and chondrogenic lineages and the expression of markers CD105(+) , CD44(+) , CD14(-) , CD34(-) , CD45(-) , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6 and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. This article is protected by copyright. All rights reserved.

  16. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling.

    PubMed

    Shih, Yu-Ru V; Hwang, YongSung; Phadke, Ameya; Kang, Heemin; Hwang, Nathaniel S; Caro, Eduardo J; Nguyen, Steven; Siu, Michael; Theodorakis, Emmanuel A; Gianneschi, Nathan C; Vecchio, Kenneth S; Chien, Shu; Lee, Oscar K; Varghese, Shyni

    2014-01-21

    Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.

  17. Effects of ketogenic diet on electroconvulsive threshold and brain contents of adenosine nucleotides.

    PubMed

    Nakazawa, M; Kodama, S; Matsuo, T

    1983-01-01

    The anticonvulsive effect of a ketogenic diet was investigated using mice fed on a ketogenic milk powder which contained medium-chain triglycerides (MCT). Electroconvulsive shocking and determination of adenosine nucleotides in mice brain were performed on three mice groups, (1) a control group; free access to a commercially available diet, (2) a fasted group; fasted for two days, and (3) a ketotic group; fasted for two days and then fed on the ketogenic milk powder for two weeks. The maximal electroconvulsive threshold of the ketotic group was significantly higher than that of the fasted group (p less than 0.001). The maximal electroconvulsive threshold of the fasted group was significantly higher than that of the control group (p less than 0.05). The contents of adenosine triphosphate (ATP) in the brain of the ketotic group was significantly higher than that of the control group (p less than 0.01). These results suggest that chronic ketosis with the ketogenic diet increases the contents of ATP in the brain and this increase in ATP probably accounts for the neuronal stability.

  18. Two strategies for the synthesis of the biologically important ATP analogue ApppI, at a multi-milligram scale

    PubMed Central

    Weisell, Janne; Vepsäläinen, Jouko

    2015-01-01

    Summary Two strategies for the synthesis of the ATP (adenosine triphosphate) analogue ApppI [1-adenosin-5’-yl 3-(3-methylbut-3-enyl)triphosphoric acid diester] (1) are described. ApppI is an active metabolite of the mevalonate pathway and thus is of major biological significance. Chemically synthezised ApppI was purified by using triethylammonium bicarbonate as the counter ion in ion-pair chromatography and characterized by 1H, 13C, 31P NMR and MS spectroscopical methods. PMID:26664641

  19. Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites

    PubMed Central

    1993-01-01

    Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6- trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP- ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously

  20. Biological effects of exogenous adenosine 5 prime -triphosphate on cultured mammalian cells: Evidence for a receptor mechanism and its regulation by desensitization

    SciTech Connect

    Gonzalez, F.A.

    1989-01-01

    Exogenous adenosine 5{prime}-triphosphate (ATP) mobilized intracellular calcium in human carcinoma A43l cells and in Swiss 3T3 and 3T6 mouse fibroblasts by increasing inositol trisphosphate similar to well down growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bradykinin (BK), serum). Calcium mobilization was examined by video imaging of fura-2 fluorescence is single cells, following the radioactive isotope {sup 45}Ca, and monitoring the decrease in fluorescence of cells loaded with chlortetracycline. Uridine 5{prime}-triphosphate, but not other nucleotides, mimicked ATP. Single-cell analysis revealed synchronous responses in 10 sec to ATP, BK or serum, while PDGF (3T3) and EGF (A431) produced slower signals with significant cell-to-cell variation. PDGF desensitized 3T3 cells to ATP and BK added 100 sec later but ATP or BK did not desensitized to PDGF. Homologous desensitization was seen with all agonists. Heterologous desensitization was also observed in A431 cells where ATP desensitized to serum, but serum did not to ATP. ATP-stimulated calcium entry was detected after 10 sec in A431 cells, but not in Swiss 3T6 cells. Entry started before significant efflux had occurred and did not fit the capacitance model of Putney. A 2-3 hr ATP pretreatment produced a homologous desensitization state that required 20 hr to disappear, probably due to down-regulation of the putative ATP receptors.

  1. Halogenated pyrrolopyrimidine analogues of adenosine from marine organisms: pharmacological activities and potent inhibition of adenosine kinase.

    PubMed

    Davies, L P; Jamieson, D D; Baird-Lambert, J A; Kazlauskas, R

    1984-02-01

    Two novel halogenated pyrrolopyrimidine analogues of adenosine, isolated from marine sources, have been examined for pharmacological and biochemical activities. 4-Amino-5-bromo-pyrrolo[2,3-d]pyrimidine, from a sponge of the genus Echinodictyum, had bronchodilator activity at least as potent as theophylline but with a different biochemical profile; unlike theophylline it had no antagonist activity at CNS adenosine receptors and it was quite a potent inhibitor of adenosine uptake and adenosine kinase in brain tissue. 5'-Deoxy-5-iodotubercidin, isolated from the red alga Hypnea valentiae, caused potent muscle relaxation and hypothermia when injected into mice. This compound was a very potent inhibitor of adenosine uptake into rat and guinea-pig brain slices and an extremely potent inhibitor of adenosine kinase from guinea-pig brain and rat brain and liver. Neither of these two pyrrolopyrimidine analogues was a substrate for, or an inhibitor of, adenosine deaminase. Neither compound appeared to have any direct agonist activity on guinea-pig brain adenosine-stimulated adenylate cyclase (A2 adenosine receptors). 5'-Deoxy-5-iodotubercidin is unique in two respects: it appears to be the first naturally-occurring example of a 5'-deoxyribosyl nucleoside and is the first example of a specifically iodinated nucleoside from natural sources. It may be the most potent adenosine kinase inhibitor yet described and, by virtue of its structure, may prove to be the most specific.

  2. Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase.

    PubMed

    Diógenes, Maria José; Neves-Tomé, Raquel; Fucile, Sergio; Martinello, Katiuscia; Scianni, Maria; Theofilas, Panos; Lopatár, Jan; Ribeiro, Joaquim A; Maggi, Laura; Frenguelli, Bruno G; Limatola, Cristina; Boison, Detlev; Sebastião, Ana M

    2014-01-01

    Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

  3. Mechanisms of ATP Release by Human Trabecular Meshwork Cells, the Enabling Step in Purinergic Regulation of Aqueous Humor Outflow

    PubMed Central

    LI, ANG; LEUNG, CHI TING; PETERSON-YANTORNO, KIM; STAMER, W. DANIEL; MITCHELL, CLAIRE H.; CIVAN, MORTIMER M.

    2011-01-01

    Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A1 adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X7 receptors (P2RX7) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically-defined contribution of PX1 and enhanced those of Cx and P2RX7. ATP release was also triggered by raising intracellular Ca2+ activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX7 antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X7 receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca2+ in TM cells, but can be modulated by oxidation-reduction state. The P2RX7-dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels. PMID:21381023

  4. Adenosine A1 receptor: Functional receptor-receptor interactions in the brain

    PubMed Central

    Sichardt, Kathrin

    2007-01-01

    Over the past decade, many lines of investigation have shown that receptor-mediated signaling exhibits greater diversity than previously appreciated. Signal diversity arises from numerous factors, which include the formation of receptor dimers and interplay between different receptors. Using adenosine A1 receptors as a paradigm of G protein-coupled receptors, this review focuses on how receptor-receptor interactions may contribute to regulation of the synaptic transmission within the central nervous system. The interactions with metabotropic dopamine, adenosine A2A, A3, neuropeptide Y, and purinergic P2Y1 receptors will be described in the first part. The second part deals with interactions between A1Rs and ionotropic receptors, especially GABAA, NMDA, and P2X receptors as well as ATP-sensitive K+ channels. Finally, the review will discuss new approaches towards treating neurological disorders. PMID:18404442

  5. A multifunctional label-free electrochemical impedance biosensor for Hg(2+), adenosine triphosphate and thrombin.

    PubMed

    Chen, Lifen; Chen, Zhong-Ning

    2015-01-01

    A multifunctional label-free biosensor for the detection of Hg(2+), adenosine triphosphate and thrombin has been developed based on the changing of the electrochemical impedance spectroscopy (EIS) from the modified electrodes when nucleic acid subunits interacting with different targets. The modified electrode consists of three interaction sections, including DNA with T-T mismatch recognizing Hg(2+) to form T-Hg(2+)-T complex, split DNA chip against ATP, and DNA domin against thrombin to form G-quadruplex. Upon DNA interaction with thrombin or ATP, an increased charge transfer resistance (Rct) had been detected. However, a decreased Rct against Hg(2+) was obtained. The Rct difference (ΔRct) has relationship with the concentration of the different targets, Hg(2+), ATP and thrombin can be selectively detected with the detection limit of 0.03, 0.25, and 0.20 nmol L(-1), respectively. To separately detect the three analytes existing in the same sample, ATP aptamer, G-rich DNA strands and EDTA were applied to mask ATP, Hg(2+) or thrombin separately.

  6. Digoxin and Adenosine Triphosphate Enhance the Functional Properties of Tissue-Engineered Cartilage

    PubMed Central

    Makris, Eleftherios A.; Huang, Brian J.; Hu, Jerry C.; Chen-Izu, Ye

    2015-01-01

    Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca2+-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca2+ modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10–14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52–110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca2+ imaging experiments revealed that both digoxin and ATP were able to increase Ca2+ oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca2+ modulators. PMID:25473799

  7. Digoxin and adenosine triphosphate enhance the functional properties of tissue-engineered cartilage.

    PubMed

    Makris, Eleftherios A; Huang, Brian J; Hu, Jerry C; Chen-Izu, Ye; Athanasiou, Kyriacos A

    2015-03-01

    Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca(2+)-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca(2+) modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10-14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52-110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca(2+) imaging experiments revealed that both digoxin and ATP were able to increase Ca(2+) oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca(2+) modulators.

  8. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge

    PubMed Central

    Kornerup, Kristin N; Page, Clive P; Moffatt, James D

    2005-01-01

    The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (ISC) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (ΔISC 69.5±6.7 μA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in ISC, which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM–10 μM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxoamido adenosine; CGS 21680; 0.1–100 μM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide; IB-MECA; 30 nM–100 μM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM–1 μM) caused a rightward shift of the adenosine concentration–effect curve only at 1 μM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration–effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline ISC was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the ISC response to adenosine in the mouse trachea. This receptor does not appear to be

  9. Pharmacological characterisation of the adenosine receptor mediating increased ion transport in the mouse isolated trachea and the effect of allergen challenge.

    PubMed

    Kornerup, Kristin N; Page, Clive P; Moffatt, James D

    2005-04-01

    The effect of adenosine on transepithelial ion transport was investigated in isolated preparations of murine trachea mounted in Ussing chambers. The possible regulation of adenosine receptors in an established model of allergic airway inflammation was also investigated. Mucosally applied adenosine caused increases in short-circuit current (I(SC)) that corresponded to approximately 50% of the response to the most efficacious secretogogue, ATP (delta I(SC) 69.5 +/- 6.7 microA cm2). In contrast, submucosally applied adenosine caused only small (<20%) increases in I(SC), which were not investigated further. The A1-selective (N6-cyclopentyladenosine, CPA, 1 nM-10 microM), A2A-selective (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxoamido adenosine; CGS 21680; 0.1-100 microM) and A3-selective (1-deoxy-1-[6-[[(3-iodophenyl)-methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide; IB-MECA; 30 nM-100 microM) adenosine receptor agonists were either equipotent or less potent than adenosine, suggesting that these receptors do not mediate the response to adenosine. The A1 receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM-1 microM) caused a rightward shift of the adenosine concentration-effect curve only at 1 microM. The mixed A2A/A2B receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) also caused rightward shift of the adenosine concentration-effect curve, again only at micromolar concentrations, suggestive of the involvement of A2B receptors. In preparations from animals sensitised to ovalbumin and challenged over 3 days with aerosol ovalbumin, a decrease in baseline I(SC) was observed and responses to ATP were diminished. Similarly, the amplitude of responses to adenosine were attenuated although there was no change in potency. These results suggest that the A2B receptor mediates the I(SC) response to adenosine in the mouse trachea. This receptor does not appear to be

  10. [Use of the bioluminescent method for the determination of bacterial adenosinetriphosphate (ATP-metry) in microbiology].

    PubMed

    Kuzikov, A N; Bondarenko, V M; Latkin, A T

    2003-01-01

    The attention of a wide circle of specialists has recently been attracted by different methods for rapid determination of pathogenic microorganisms in biological specimens, environmental objects and foodstuffs, as well as in cases of possible acts of bioterrorism. In this respect the bioluminescent method for determination of adenosine triphosphate (ATP) contained in microbial cells is of interest. The method is based on the interaction ATP, luciferase and luciferin, accompanied by giving off energy in the form of light emission. When compared with routine methods, the use of this method considerably reduces the duration of the analysis, and its high sensitivity is comparable with that of the polymerase chain reaction. In this review the data on the prospects of the practical use of the bioluminescent method of ATP-metry are presented.

  11. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

    Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

  12. Repeated administration of adenosine increases its cardiovascular effects in rats.

    PubMed

    Vidrio, H; García-Márquez, F; Magos, G A

    1987-01-20

    Hypotensive and negative chronotropic responses to adenosine in anesthetized rats increased after previous administration of the nucleoside. Bradycardia after adenosine in the isolated perfused rat heart was also potentiated after repeated administration at short intervals. This self-potentiation could be due to extracellular accumulation of adenosine and persistent stimulation of receptors caused by saturation or inhibition of cellular uptake of adenosine.

  13. Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

    USGS Publications Warehouse

    Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

    2009-01-01

    Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

  14. Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

    PubMed

    Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

    2007-09-01

    beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

  15. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

    PubMed Central

    Chen, Daniel T.N.; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-01-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme’s ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 105 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies. PMID:26682814

  16. Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain.

    PubMed

    Hanna, Eissa; Ng, Kit Fai; MacRae, Ian J; Bley, Christopher J; Fisher, Andrew J; Segel, Irwin H

    2004-02-06

    ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.

  17. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Chen, Daniel T. N.; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-01

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights into the beating mechanism of flagella and a powerful tool for future studies.

  18. ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level.

    PubMed

    Chen, Daniel T N; Heymann, Michael; Fraden, Seth; Nicastro, Daniela; Dogic, Zvonimir

    2015-12-15

    The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.

  19. Conformational Changes Produced by ATP Binding to the Plasma Membrane Calcium Pump*

    PubMed Central

    Mangialavori, Irene C.; Ferreira-Gomes, Mariela S.; Saffioti, Nicolás A.; González-Lebrero, Rodolfo M.; Rossi, Rolando C.; Rossi, Juan Pablo F. C.

    2013-01-01

    The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only. PMID:24025327

  20. Conformational changes produced by ATP binding to the plasma membrane calcium pump.

    PubMed

    Mangialavori, Irene C; Ferreira-Gomes, Mariela S; Saffioti, Nicolás A; González-Lebrero, Rodolfo M; Rossi, Rolando C; Rossi, Juan Pablo F C

    2013-10-25

    The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

  1. Application of bioluminescence ATP measurement for evaluation of fungal viability of foxing spots on old documents.

    PubMed

    Rakotonirainy, Malalanirina Sylvia; Dubar, Pauline

    2013-01-01

    An adenosine triphosphate (ATP) bioluminescence-based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish-red stains, damaging the aesthetics of documents and their long-term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold-like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm(2), suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm(2). ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots.

  2. The use of adenosine and adenosine triphosphate testing in the diagnosis, risk stratification and management of patients with syncope: current evidence and future perspectives.

    PubMed

    Fragakis, Nikolaos; Antoniadis, Antonios P; Saviano, Massimo; Vassilikos, Vassilios; Pappone, Carlo

    2015-03-15

    Syncope is a significant source of cardiovascular-related morbidity yet the etiology is frequently obscure and the identification of patients at highest risk is challenging. Adenosine (AD) and adenosine triphosphate (ATP) administrations have been suggested as potentially useful non-invasive tools in the diagnostic workup of patients with neurally-mediated or bradycardia-related syncope. It has been postulated that both compounds by modulating the autonomic innervation in the heart and exerting negative chronotropic and dromotropic effects in the conduction system, may unmask the mechanism of syncope. However, the clinical implications derived from the efficacy of both tests in the investigation of syncope remain unclear mainly due to inconclusive and occasionally contradictory results of published studies. This review article summarizes recent and past information in the use of ATP and AD in the investigation of syncope with emphasis on clinical trials. We present the current level of evidence for the use of these agents in clinical practice, identify areas where further research is warranted and highlight the future perspectives of these agents as complements to an accurate risk-stratification of patients with syncope.

  3. Adenosine receptors as drug targets — what are the challenges?

    PubMed Central

    Chen, Jiang-Fan; Eltzschig, Holger K.; Fredholm, Bertil B.

    2014-01-01

    Adenosine signalling has long been a target for drug development, with adenosine itself or its derivatives being used clinically since the 1940s. In addition, methylxanthines such as caffeine have profound biological effects as antagonists at adenosine receptors. Moreover, drugs such as dipyridamole and methotrexate act by enhancing the activation of adenosine receptors. There is strong evidence that adenosine has a functional role in many diseases, and several pharmacological compounds specifically targeting individual adenosine receptors — either directly or indirectly — have now entered the clinic. However, only one adenosine receptor-specific agent — the adenosine A2A receptor agonist regadenoson (Lexiscan; Astellas Pharma) — has so far gained approval from the US Food and Drug Administration (FDA). Here, we focus on the biology of adenosine signalling to identify hurdles in the development of additional pharmacological compounds targeting adenosine receptors and discuss strategies to overcome these challenges. PMID:23535933

  4. Cold induced changes of adenosine levels in common eelpout (Zoarces viviparus): a role in modulating cytochrome c oxidase expression.

    PubMed

    Eckerle, L G; Lucassen, M; Hirse, T; Pörtner, H O

    2008-04-01

    Exposure of ectothermic organisms to variations in temperatures causes a transient mismatch between energy supply and demand, which needs to be compensated for during acclimation. Adenosine accumulation from ATP breakdown indicates such an imbalance and its reversal reflects a restoration of energy status. We monitored adenosine levels in blood serum and liver of common eelpout (Zoarces viviparus) during cold exposure in vivo. Furthermore, we tested its effect on the pattern of thermal acclimation in hepatocytes isolated from cold- (4 degrees C) versus warm- (11 degrees C) exposed fish. Adenosine levels increased during cold exposure in vivo and reached a transient maximum after 24 h in serum, but remained permanently elevated in liver. Whole animal cold acclimation induced a rise of liver citrate synthase activity by 44+/-15%, but left cytochrome c oxidase activity (COX) and RNA expression of the respective genes unchanged. Cold incubation of hepatocytes from warm-acclimated fish failed to cause an increase of mitochondrial enzyme activities despite increased COX4 mRNA levels. Conversely, warm acclimation of hepatocytes from cold-acclimated fish reduced both enzyme activities and COX2 and COX4 mRNA levels by 26-37%. Adenosine treatment of both warm- and cold-acclimated hepatocytes suppressed COX activities but activated COX mRNA expression. These effects were not receptor mediated. The present findings indicate that adenosine has the potential to regulate mitochondrial functioning in vivo, albeit the pathways resulting in the contrasting effects on expression and activity need to be identified.

  5. Enhanced Diffusion of Molecular Motors in the Presence of Adenosine Triphosphate and External Force

    NASA Astrophysics Data System (ADS)

    Shinagawa, Ryota; Sasaki, Kazuo

    2016-06-01

    The diffusion of a molecular motor in the presence of a constant external force is considered on the basis of a simple theoretical model. The motor is represented by a Brownian particle moving in a series of parabolic potentials placed periodically on a line, and the potential is switched stochastically from one parabola to another by a chemical reaction, which corresponds to the hydrolysis or synthesis of adenosine triphosphate (ATP) in motor proteins. It is found that the diffusion coefficient as a function of the force exhibits peaks. The mechanism of this diffusion enhancement and the possibility of observing it in F1-ATPase, a biological rotary motor, are discussed.

  6. ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo-osmotic shock.

    PubMed

    Azorin, Nathalie; Raoux, Matthieu; Rodat-Despoix, Lise; Merrot, Thierry; Delmas, Patrick; Crest, Marcel

    2011-05-01

    Touch is detected through receptors located in the skin and the activation of channels in sensory nerve fibres. Epidermal keratinocytes themselves, however, may sense mechanical stimulus and contribute to skin sensation. Here, we showed that the mechanical stimulation of human keratinocytes by hypo-osmotic shock releases adenosine triphosphate (ATP) and increases intracellular calcium. We demonstrated that the release of ATP was found to be calcium independent because emptying the intracellular calcium stores did not cause ATP release; ATP release was still observed in the absence of external calcium and it persisted on chelating cytosolic calcium. On the other hand, the released ATP activated purinergic receptors and mobilized intracellular calcium stores. The resulting depletion of stored calcium led to the activation of capacitative calcium entry. Increase in cytosolic calcium concentration was blocked by the purinergic receptor blocker suramin, phospholipase C inhibitor and apyrase, which hydrolyses ATP. Collectively, our data demonstrate that human keratinocytes are mechanically activated by hypo-osmotic shock, leading first to the release of ATP, which in turn stimulates purinergic receptors, resulting in the mobilization of intracellular calcium and capacitative calcium entry. These results emphasize the crucial role of ATP signalling in the transduction of mechanical stimuli in human keratinocytes.

  7. In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.

    PubMed

    Takahashi, Toshiya; Kimura, Yutaka; Niwa, Kazuki; Ohmiya, Yoshihiro; Fujimura, Taku; Yamasaki, Kenshi; Aiba, Setsuya

    2013-10-01

    Adenosine 5'-triphosphate (ATP) release from keratinocytes has been observed in various stress models in vitro, but studies demonstrating epidermal ATP release in vivo are limited. To visualize extracellular ATP (eATP) in vivo, we developed enhanced green-emitting luciferase immobilized on agarose beads (Eluc-agarose). Subcutaneous injection of Eluc-agarose together with ATP into the dorsal skin of BALB/c mice following intraperitoneal luciferin injection produced detectable and measurable bioluminescence using an in vivo imaging system. Using Eluc-agarose, we demonstrated in vivo that bright bioluminescence was observed from 1 to 20 minutes after repeated tape stripping of murine skin. This bioluminescence was suppressed by the local administration of apyrase. Eluc-agarose bioluminescence was observed only in tape-stripped skin with transepidermal water loss (TEWL) between 100 and 140 g m(2) h(-1), indicating a loss of bioluminescence with excessive tape stripping (TEWL>140 g m(-2) h(-1)). Histologically, tape-stripped skin with detectable eATP had a viable epidermis and a subepidermal neutrophil infiltrate, and administration of apyrase reduced the inflammatory infiltrate. Neither a viable epidermis nor an upper dermal neutrophil infiltrate was observed after excessive tape stripping. These results suggest that tape stripping prompts ATP release from viable keratinocytes, which facilitates inflammatory cell migration. Eluc-agarose may be useful in the in vivo detection of eATP in murine models of skin diseases.

  8. Can measuring environmental cleanliness using ATP aid in the monitoring of wards with periods of increased incidence of Clostridium difficile?

    PubMed Central

    Hardy, Katherine; Abbott, Gill; Bashford, Sarah; Bucior, Helen; Codd, Jane; Holland, Madelaine; Reynolds, Mandy; Simms, Avril

    2013-01-01

    Management of periods of increased incidence of Clostridium difficile (PIIs) on a ward have become multi-factorial and involve isolation of patients, typing of the isolates, antibiotic audit and a weekly environmental audit completed until three consecutive weekly passes are obtained. The aim of this study was to establish if monitoring the environment using adenosine triphosphate (ATP) could aid in reducing the length of time the wards remained on the weekly environmental audit. Secondly, it was to establish if certain pieces of equipment had continually high ATP scores requiring wider interventions. The study took place across three hospital sites covered by one infection control team over a 22 month period. There were three study periods, with the only difference being that ATP monitoring was conducted during period B. There was a difference in the length of time the wards remained on the audit between the first period and the ATP period; however this decrease was sustained in the third period when ATP monitoring ceased. There was an increase in the percentage of sites achieving a pass with ATP week on week. ATP monitoring provided the staff with non-subjective results and immediate feedback that facilitated discussions about cleaning regimes. ATP monitoring was a useful adjunct to environmental audits.

  9. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    PubMed

    Pérez-Aguilar, Mary Carmen; Rondón-Mercado, Rocío

    2015-09-01

    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies.

  10. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... This Page Bras J, Guerreiro R, Santo GC. Mutant ADA2 in vasculopathies. N Engl J Med. 2014 ... M, Anikster Y, King MC, Levy-Lahad E. Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy. ...

  11. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

    Holtzman, S.G.; Mante, S.; Minneman, K.P. )

    1991-01-01

    Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity of caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity.

  12. Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

    PubMed

    Brozmanova, M; Mazurova, L; Ru, F; Tatar, M; Hu, Y; Yu, S; Kollarik, M

    2016-02-01

    Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

  13. A Destabilized Case of Stable Effort Angina Pectoris Induced by Low-dose Adenosine Triphosphate.

    PubMed

    Sueta, Daisuke; Kojima, Sunao; Izumiya, Yasuhiro; Yamamuro, Megumi; Kaikita, Koichi; Hokimoto, Seiji; Ogawa, Hisao

    A 79-year-old man was diagnosed with sudden deafness. He had previously experienced a suspected episode of angina pectoris. At a local hospital, after 500 mg of hydrocortisone and 80 mg adenosine triphosphate (ATP) were administered, he became aware of chest discomfort. An electrocardiogram revealed serious ST-segment depressions. He was diagnosed with a non-ST elevated myocardial infarction (NSTEMI). Emergency coronary angiography revealed triple vessel disease, and the lesion was successfully stented. The mechanisms whereby the stable effort angina pectoris destabilized in this case were thought to include a reduction of the local blood flow because of an ATP product and probable thrombus formation in response to the administered steroids.

  14. A Destabilized Case of Stable Effort Angina Pectoris Induced by Low-dose Adenosine Triphosphate

    PubMed Central

    Sueta, Daisuke; Kojima, Sunao; Izumiya, Yasuhiro; Yamamuro, Megumi; Kaikita, Koichi; Hokimoto, Seiji; Ogawa, Hisao

    2016-01-01

    A 79-year-old man was diagnosed with sudden deafness. He had previously experienced a suspected episode of angina pectoris. At a local hospital, after 500 mg of hydrocortisone and 80 mg adenosine triphosphate (ATP) were administered, he became aware of chest discomfort. An electrocardiogram revealed serious ST-segment depressions. He was diagnosed with a non-ST elevated myocardial infarction (NSTEMI). Emergency coronary angiography revealed triple vessel disease, and the lesion was successfully stented. The mechanisms whereby the stable effort angina pectoris destabilized in this case were thought to include a reduction of the local blood flow because of an ATP product and probable thrombus formation in response to the administered steroids. PMID:27853071

  15. Erythrocyte 2,3-diphosphoglycerate and adenosine-triphosphate in cretins living at high altitude.

    PubMed

    Adams, W H

    1976-01-01

    A comparison of concentrations of 2,3-diphosphoglycerate (2,3-DPG) and adenosine-triphosphate (ATP) in the red cells of cretins and normal controls living at 3,700 m in the Nepal Himalayas has shown that 2,3-DPG and ATP levels were higher in the cretins. A negative correlation between hemoglobin and 2.3-DPG level was found. Chronic hypoxia appears to have provided the additional stress required to differentiate the significance of thyroid hormone deficiency in producing anemia from its effect on 2,3-DPG levels. If thyroid hormone is in fact one regulator of 2,3-DPG, the anemia of hypothyroidism appears to be more significant. This also suggest that the anemia of hypothyroidism, is at least in part, "pathologic" as opposed to "adaptive".

  16. Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.

    PubMed Central

    Pollard, T D; Bhandari, D; Maupin, P; Wachsstock, D; Weeds, A G; Zot, H G

    1993-01-01

    We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not

  17. Sensitive fluorescence detection of ATP based on host-guest recognition between near-infrared β-Cyclodextrin-CuInS2 QDs and aptamer.

    PubMed

    Hu, Tianyu; Na, Weidan; Yan, Xu; Su, Xingguang

    2017-04-01

    We have developed a near-infrared (NIR) fluorescent aptamer-based sensor for sensitive detection of adenosine-5'-triphosphate (ATP) by using a ATP-binding aptamer and β-Cyclodextrin-CuInS2 quantum dots (β-CD-CuInS2 QDs). The fluorescence of β-CD-CuInS2 QDs has a slight enhancement with the addition of ATP-binding aptamer due to the host-guest recognition between aptamer and β-CD. When ATP is added, it will bind to aptamer to form G-quadruplexes. Aptamer-ATP complexes can enter into the hydrophobic cavities of β-CD and result in great enhancement of the fluorescence intensity. Under the optimum conditions, the fluorescence intensity of β-CD-CuInS2 QDs is proportional to the concentration of ATP, which shows a good linear response toward ATP concentration range of 6-1200μmolL(-1), the detection limit for ATP is 3μmolL(-1). The present assay shows a good selectivity for ATP over other biologically important proteins, and it is applied to the determination of ATP in human serum sample with satisfactory results.

  18. Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut

    PubMed Central

    Durnin, Leonie; Hwang, Sung Jin; Kurahashi, Masaaki; Drumm, Bernard T.; Ward, Sean M.; Sasse, Kent C.; Sanders, Kenton M.; Mutafova-Yambolieva, Violeta N.

    2014-01-01

    Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD+ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca2+-activated small-conductance K+ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα+ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca2+ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD+, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon. PMID:25341729

  19. Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis.

    PubMed

    Sperlágh, B; Mergl, Z; Jurányi, Z; Vizi, E S; Makara, G B

    1999-03-01

    It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.

  20. Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase.

    PubMed

    Kuby, S A; Palmieri, R H; Frischat, A; Fischer, A H; Wu, L H; Maland, L; Manship, M

    1984-05-22

    The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. DNA aptasensor for the detection of ATP based on quantum dots electrochemiluminescence

    NASA Astrophysics Data System (ADS)

    Huang, Haiping; Tan, Yanglan; Shi, Jianjun; Liang, Guoxi; Zhu, Jun-Jie

    2010-04-01

    A novel and facile strategy for the fabrication of aptamer-based adenosine 5'-triphosphate (ATP) biosensor was developed by a quantum dot (QD) electrochemiluminescence (ECL) technique. Different from the existing strategies for the development of aptasensors based on electrochemical, fluorescent or other methods, the strategy proposed here is essentially based on the aptamer-ATP specific affinity and the rules of Watson-Crick base pairing. After the thiol modified anti-ATP probes were immobilized onto the pretreated Au electrode, the electrode was incubated in ATP solution to form aptamer-ATP bioaffinity complexes. The complementary DNA (cDNA) oligonucleotides were hybridized with the free probes. As a result, the avidin-modified QDs were bound to the aptasensor through the biotin-avidin system in the existence of biotin-modified cDNA. The ECL signal of the aptasensor was responsive to the amount of QDs bound to the cDNA oligonucleotides, which was inversely proportional to the combined target analyte ATP. The QDs were characterized by high resolution transmission electron microscopy (HRTEM), ultraviolet (UV) and photoluminescence (PL) spectra. The preparation process for the aptasensor was monitored by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Possible interference, such as from the pH value of the electrolyte, the incubation time and the concentration of coreactant K2S2O8, on the aptasensor ECL response were investigated. The ATP concentration was measured through the decrease of ECL intensity. The ECL intensity of the aptasensor decreased with the increase of the logarithm of the ATP concentration over the 0.018-90.72 μM range. In addition, the aptasensor exhibited excellent selectivity responses toward the target analyte. This study may offer a new and relatively general approach to expand the application of QD ECL in the aptasensor field.

  2. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium

    SciTech Connect

    Pratt, A.D.; Clancy, G.; Welsh, M.J.

    1986-08-01

    Adenosine is a local regulator of a variety of physiological functions in many tissues and has been observed to stimulate secretion in several Cl-secreting epithelia. In canine tracheal epithelium the authors found that adenosine stimulates Cl secretion from both the mucosal and submucosal surfaces. Addition of adenosine, or its analogue 2-chloroadenosine, to the mucosal surface potently stimulated Cl secretion with no effect on the rate of Na absorption. Stimulation resulted from an interaction of adenosine with adenosine receptors, because it was blocked by the adenosine receptor blocker, 8-phenyltheophylline. The adenosine receptor was a stimulatory receptor as judged by the rank-order potency of adenosine and its analogues and by the increase in cellular adenosine 3',5'-cyclic monophosphate levels produced by 2-chloroadenosine. Adenosine also stimulated Cl secretion when it was added to the submucosal surface, although the maximal increase in secretion was less and it was much less potent. The observation that mucosal 8-phenyletheophylline blocked the effect of submucosal 2-chloroadenosine, whereas submucosal 8-phenyltheophylline did not prevent a response to mucosal or submucosal 2-chloroadenosine, suggests that adenosine receptors are located on the mucosal surface. Thus submucosal adenosine may stimulate secretion by crossing the epithelium and interacting with receptors located on the mucosal surface. Because adenosine can be released from mast cells located in the airway lumen in response to inhaled material, and because adenosine stimulated secretion from the mucosal surface, it may be in a unique position to control the epithelium on a regional level.

  3. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    PubMed

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  4. Neurabin scaffolding of adenosine receptor and RGS4 regulates anti-seizure effect of endogenous adenosine.

    PubMed

    Chen, Yunjia; Liu, Yin; Cottingham, Christopher; McMahon, Lori; Jiao, Kai; Greengard, Paul; Wang, Qin

    2012-02-22

    Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A(1) receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G-protein signaling 4 (RGS4), a protein known to turn off G-protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ∼50% rate of death in wild-type (WT) mice did not affect neurabin-null mice or WT mice cotreated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

  5. RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX.

    PubMed

    Burke, D H; Gold, L

    1997-05-15

    We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.

  6. Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin.

    PubMed

    Chen, Jinyang; Liu, Yucheng; Ji, Xinghu; He, Zhike

    2016-09-15

    In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5'-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3'-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design.

  7. ATP bioluminescence rapid detection of total viable count in soy sauce.

    PubMed

    Yan, Shou-Lei; Miao, Su-Na; Deng, Shao-Ya; Zou, Min-Juan; Zhong, Fo-Sheng; Huang, Wen-Biao; Pan, Si-Yi; Wang, Qing-Zhang

    2012-01-01

    The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10(2) -3 × 10(4) CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB-941) and 96-well plates and could analyse 50-100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce.

  8. Estimation of PMI depends on the changes in ATP and its degradation products.

    PubMed

    Mao, Shiwei; Fu, Gaowen; Seese, Ronald R; Wang, Zhen-Yuan

    2013-09-01

    Estimating the time since death, or postmortem interval (PMI), has been one of the biggest difficulties in modern forensic investigation. This study tests if the concentrations of breakdown products of adenosine triphosphate (ATP) correlate with PMI in multiple organs from rat. Brains, spleens, and kidneys of rats were harvested at different time points in carcasses maintained at 4°C or 20°C. High Performance Liquid Chromatography (HPLC) was used to quantify concentrations of metabolites related to ATP degradation. A K value (Kv=100×(Hx+HxR)/(ATP+ADP+AMP+IMP+HxR+Hx)) was calculated and correlated with PMI for each organ and temperature. The results indicate that the K value is a robust index for the estimation of PMI based on highly significant linear correlations between PMI and concentrations of ATP breakdown products. Compared with other current research methods, the changing tendency of ATP and its degradation products may be potentially a better way for the estimation of PMI in medico-legal practice.

  9. Metabolic networks to generate pyruvate, PEP and ATP from glycerol in Pseudomonas fluorescens.

    PubMed

    Alhasawi, Azhar; Thomas, Sean C; Appanna, Vasu D

    2016-04-01

    Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H2O2) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O2-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H2O2-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.

  10. Bidirectional astrocyte-neuron communication: the many roles of glutamate and ATP.

    PubMed

    Fellin, Tommaso; Sul, Jai-Yoon; D'Ascenzo, Marcello; Takano, Hajime; Pascual, Olivier; Haydon, Philip G

    2006-01-01

    Glutamatergic and purinergic signalling play key roles in synaptic transmission and modulation in the CNS. Here, we review recent evidence showing that glial cells, and in particular astrocytes, are active players in ATP and glutamate signalling in the brain. ATP and glutamate coordinately activate astrocytes, through the mobilization of their internal Ca2+, which in turn triggers the release from astrocytes of several neuroactive molecules including ATP and glutamate themselves. These 'gliotransmitters' signal either to astrocytes, where they generate Ca2+ waves, or to neurons, where they modulate synaptic transmission and neuronal excitability. By using microfabricated lanes of adhesive substrate, we provide further evidence for a diffusible factor-mediated propagation of Ca2+ waves and, through flash photolysis experiments in hippocampal slices, we show that glutamate and ATP cooperate in the generation of the astrocytic Ca2+ signal. Once astrocytes are activated they provide both excitatory and inhibitory effects on neighbouring neurons. Through the Ca2+-dependent release of glutamate, which acts on extrasynaptic neuronal NMDA receptors, astrocytes excite neurons while, in contrast, ATP released from astrocytes, after the delayed conversion to adenosine, causes neuronal suppression.

  11. ATP induces NO production in hippocampal neurons by P2X(7) receptor activation independent of glutamate signaling.

    PubMed

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3')-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N(ω)-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

  12. Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

    PubMed

    Leustek, T; Murillo, M; Cervantes, M

    1994-07-01

    ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).

  13. ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

    PubMed Central

    Codocedo, Juan Francisco; Godoy, Juan Alejandro; Poblete, Maria Ines; Inestrosa, Nibaldo C.; Huidobro-Toro, Juan Pablo

    2013-01-01

    To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity. PMID:23472093

  14. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  15. Radioimmunochemical quantitation of human adenosine deaminase.

    PubMed Central

    Daddona, P E; Frohman, M A; Kelley, W N

    1979-01-01

    Markedly reduced or absent adenosine deaminase activity in man is associated with an autosomal recesive form of severe conbined immunodeficiency disease. To further define the genetic nature of this enzyme defect, we have quantitated immunologically active adenosine deaminase (CRM) in the hemolysate of homozygous deficient patients and their heterozygous parents. A highly specific radioimmunoassay was developed capable of detecting 0.05% of normal erythrocyte adenosine deaminase. Hemolysates from nine heterozygotes (five families) showed a wide range in CRM (32--100% of normal) and variable absolute specific activities with several being at least 1 SD BELOW THE NORMAL MEAN. Hemolysates from four unrelated patients showed less than 0.09% adenosine deaminase activity with CRM ranging from less than 0.06 to 5.6% of the normal mean. In conclusion, heterozygote and homozygote hemolysates from five of the eight families analyzed revealed variable levels of CRM suggesting heterogeneous genetic alteration or expression of the silent or defective allele(s) of adenosine deaminase. PMID:468994

  16. The adenosine kinase hypothesis of epileptogenesis

    PubMed Central

    Boison, Detlev

    2008-01-01

    Current therapies for epilepsy are largely symptomatic and do not affect the underlying mechanisms of disease progression, i.e. epileptogenesis. Given the large percentage of pharmacoresistant chronic epilepsies, novel approaches are needed to understand and modify the underlying pathogenetic mechanisms. Although different types of brain injury (e.g. status epilepticus, traumatic brain injury, stroke) can trigger epileptogenesis, astrogliosis appears to be a homotypic response and hallmark of epilepsy. Indeed, recent findings indicate that epilepsy might be a disease of astrocyte dysfunction. This review focuses on the inhibitory neuromodulator and endogenous anticonvulsant adenosine, which is largely regulated by astrocytes and its key metabolic enzyme adenosine kinase (ADK). Recent findings support the “ADK hypothesis of epileptogenesis”: (i) Mouse models of epileptogenesis suggest a sequence of events leading from initial downregulation of ADK and elevation of ambient adenosine as an acute protective response, to changes in astrocytic adenosine receptor expression, to astrocyte proliferation and hypertrophy (i.e. astrogliosis), to consequential overexpression of ADK, reduced adenosine and – finally – to spontaneous focal seizure activity restricted to regions of astrogliotic overexpression of ADK. (ii) Transgenic mice overexpressing ADK display increased sensitivity to brain injury and seizures. (iii) Inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy. (iv) Intrahippocampal implants of stem cells engineered to lack ADK prevent epileptogenesis. Thus, ADK emerges both as a diagnostic marker to predict, as well as a prime therapeutic target to prevent, epileptogenesis. PMID:18249058

  17. Caffeine, adenosine receptors, and synaptic plasticity.

    PubMed

    Costenla, Ana Rita; Cunha, Rodrigo A; de Mendonça, Alexandre

    2010-01-01

    Few studies to date have looked at the effects of caffeine on synaptic plasticity, and those that did used very high concentrations of caffeine, whereas the brain concentrations attained by regular coffee consumption in humans should be in the low micromolar range, where caffeine exerts pharmacological actions mainly by antagonizing adenosine receptors. Accordingly, rats drinking caffeine (1 g/L) for 3 weeks, displayed a concentration of caffeine of circa 22 microM in the hippocampus. It is known that selective adenosine A1 receptor antagonists facilitate, whereas selective adenosine A2A receptor antagonists attenuate, long term potentiation (LTP) in the hippocampus. Although caffeine is a non-selective antagonist of adenosine receptors, it attenuates frequency-induced LTP in hippocampal slices in a manner similar to selective adenosine A2A receptor antagonists. These effects of low micromolar concentration of caffeine (30 microM) are maintained in aged animals, which is important when a possible beneficial effect for caffeine in age-related cognitive decline is proposed. Future studies will still be required to confirm and detail the involvement of A1 and A2A receptors in the effects of caffeine on hippocampal synaptic plasticity, using both pharmacological and genetic approaches.

  18. Diguanosinetetraphosphatase from rat liver: Acitivity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate.

    PubMed

    Lobatón, C D; Vallejo, C G; Sillero, A; Sillero, M A

    1975-01-15

    The hydrolysis of diadenosine tetraphosphate, a compound previously described by others to occur in liver at concentrations of around 0.1 mu M, is carried out by a specific enzyme. This enzyme has been partially purified from rat liver extracts, and the following properties have been found. The Km value for diadenosine tetraphosphate is 2 mu M; the products of hydrolysis are ATP and AMP; the Km value for diguanosine tetraphosphate is 2 mu M; none of the following substances were substrates of the enzyme: diadenosine triphosphate, diguanosine di and triphosphates, adenosine tetraphosphate, ATP, ADP, NAD+, NADP+ and bis-p-nitrophenylphosphate. Cyclic AMP was not an inhibitor of the reaction. The enzyme requires Mg2+ ions, is maximally active at a pH value of approximately 8, and has a molecular weight of 22000 as estimated by filtration on Sephadex G-100. The activation energy of the reaction was of 10250 cal times mol-1 (42886 J times mol-1). Particularly striking is the inhibition by adenosine tetraphosphate (Ki equals 48 nM) and guanosine tetraphosphate (Ki equals 14 nM). Other nucleotides tested were also competitive inhibitors with Ki values in the 10--100 mu M range.

  19. Time courses and time-resolved spectra of firefly bioluminescence initiated by two methods of ATP injection and photolysis of caged ATP.

    PubMed

    Yanagisawa, Yuki; Kageyama, Takeshi; Wada, Naohisa; Tanaka, Masatoshi; Ohno, Shin-Ya

    2013-01-01

    The time-dependent characteristics of firefly bioluminescence initiated by manual injection of adenosine triphosphate (ATP) into buffer solution containing luciferin (Ln), luciferase (Luc) and Mg(2+) were measured with a resolution of 10 ms, and compared with those obtained by photolysis of caged ATP. The time course depends on pH; both rise and decay rates decrease when pH is lowered from 7.8 to 6.8. In contrast, the parameter λ in the kinetic formula related to diffusion of ATP is almost independent of pH. The pH dependence of the time course of bioluminescence can be explained by the same pH tendency as the rate of ATP binding at the active site of Luc. The time-resolved spectra can be decomposed into two Gaussian components with maxima at 2.2 and 2.0 eV. At pH 7.8, the band at 2.2 eV is more intense than that at 2.0 eV for all three concentration conditions. At lower pH, the band at 2.2 eV becomes weaker than that at 2.0 eV. The intensity ratio of the 2.0 and 2.2 eV bands is constant for duration time of 600 s for both injection and photolysis experiments, and the above conclusions are unaffected by the concentration ratio [Ln]/[Luc].

  20. The Therapeutic Potential of Adenosine Triphosphate as an Immune Modulator in the Treatment of HIV/AIDS: A Combination Approach with HAART

    PubMed Central

    Wagner, Marc C.E.

    2011-01-01

    Extracellular adenosine triphosphate (eATP) is a potent molecule that has the capacity to modulate various aspects of cell functions including gene expression. This element of modulation is essential to the role of ATP as a therapeutic agent. The hypothesis presented is that ATP can have an important impact on the treatment of HIV infection. This is supported in part by published research, although a much greater role for ATP is suggested than prior authors ever thought possible. ATP has the ability to enhance the immune system and could thus improve the host’s own defense mechanisms to eradicate the virus-infected cells and restore normal immune function. This could provide effective therapy when used in conjunction with highly active antiretroviral therapies (HAART) to eliminate the latently infected cells. The key lies in applying ATP through the methodology described. This article presents a strategy for using ATP therapeutically along with background evidence to substantiate the importance of using ATP in the treatment of HIV infection. PMID:21675943

  1. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  2. High affinity P2x-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes.

    PubMed Central

    Michel, A. D.; Humphrey, P. P.

    1996-01-01

    1. The binding sites labelled by [35S]-adenosine 5'-O-[3-thiotriphosphate]([35S]-ATP gamma S) at 4 degrees C in rat vas deferens membranes were studied and compared to the sites labelled by [3H]-alpha,beta-methylene ATP ([3H]-alpha beta meATP) to ascertain whether [35S]-ATP gamma S can be used to label the P2x purinoceptor. 2. In the presence of 4 mM CaCl2, the binding of 0.2 nM [35S]-ATP gamma S to vas deferens membranes was increased 3.4 fold, when compared to studies performed in the absence of calcium. However, binding did not appear to be solely to P2x purinoceptors since [35S]-ATP gamma S labelled a heterogeneous population of sites and about 72% of the sites possessed high affinity (pIC50 = 7.5) for guanosine 5'-O-[3-thiotriphosphate] (GTP gamma S). Even in the presence of 1 microM GTP gamma S, to occlude the sites with high affinity for GTP gamma S, the binding of [35S]-ATP gamma S was heterogeneous and since there was also evidence of extensive metabolism of ATP in the presence of calcium, the binding of [35S]-ATP gamma S under these conditions was not studied further. 3. In the absence of calcium ions, [35S]-ATP gamma S bound to a single population of sites (pKD = 9.23; Bmax = 4270 fmol mg-1 protein). Binding reached steady state within 3 h (t1/2 = 38 min), was stable for a further 4 h and was readily reversible upon addition of 10 microM unlabelled ATP gamma S (t1/2 = 45 min). In competition studies the binding of 0.2 nM [35S]-ATP gamma S was inhibited by a number of P2x purinoceptor agonists and antagonists, but not by adenosine receptor agonists, staurosporine (1 microM) or several ATPase inhibitors. The rank order of agonist affinity estimates (pIC50 values) in competing for the [35S]-ATP gamma S binding sites was: ATP (9.01), 2-methylthio- ATP (8.79), ATP gamma S (8.73), alpha beta meATP (7.57), ADP (7.24), beta, gamma-methylene ATP (7.18), L-beta, gamma-methylene ATP (5.83), alpha, beta-methylene ADP (4.36). 4. Affinity estimates (pIC50 values) for

  3. Validation of the AccuPoint Advanced ATP Hygiene Monitoring System for Sanitation Monitoring Through Detection of ATP from Stainless Steel Surfaces.

    PubMed

    Viator, Ryan; Gray, R Lucas; Sarver, Ron; Steiner, Brent; Mozola, Mark; Rice, Jennifer

    2017-03-01

    The AccuPoint Advanced ATP Hygeine Monitoring System was validated by an AOAC International Performance Tested MethodSM on the detection of ATP from stainless steel surfaces. Neogen Corp.'s system is a lightweight, hand-held diagnostic tool used to validate and verify a hygiene program's effectiveness by detecting organic residues remaining on surfaces and in liquids after cleaning. The system is composed of three primary components: an electronic luminometer, fully self-contained single-use samplers, and software. The system is designed to detect adenosine triphosphate (ATP) at set thresholds and to report the measurement in relative light units (RLU). These thresholds are established by a facility to reflect effective cleaning practices. The instrument compares the measured level of ATP with the established threshold and reports the results as pass, marginal, or fail. A linear dose-response in RLU was observed with pure analyte. In the matrix and microbial studies, detection levels varied depending on the matrix and microorganism tested. Independent laboratory trials confirmed pure analyte and matrix observations. Specificity testing of similar, yet different, compounds resulted in 0 RLU for all except 2'-deoxyadenosine 5'-triphosphate sodium salt, which showed markedly reduced reactivity when compared with ATP. Also, interference by these compounds was negligible. When disinfectant residues were evaluated for their effect on the test, cleaners increased RLU output to varying degrees. Stability testing showed consistent results between three independently manufactured lots and stable results through the 9 month shelf-life. Additionally, when three readers were compared using electronic light-emitting diodes as the light source, instrument variability was low (<3%). Robustness testing results provided evidence that temperature affects test performance more than shaking time, and sampler performance improves as the temperature increases to room temperature. These

  4. Adenine and adenosine salvage in Leishmania donovani.

    PubMed

    Boitz, Jan M; Ullman, Buddy

    2013-08-01

    6-aminopurine metabolism in Leishmania is unique among trypanosomatid pathogens since this genus expresses two distinct routes for adenine salvage: adenine phosphoribosyltransferase (APRT) and adenine deaminase (AAH). To evaluate the relative contributions of APRT and AAH, adenine salvage was evaluated in Δaprt, Δaah, and Δaprt/Δaah null mutants of L. donovani. The data confirm that AAH plays the dominant role in adenine metabolism in L. donovani, although either enzyme alone is sufficient for salvage. Adenosine salvage was also evaluated in a cohort of null mutants. Adenosine is also primarily converted to hypoxanthine, either intracellularly or extracellularly, but can also be phosphorylated to the nucleotide level by adenosine kinase when the predominant pathways are genetically or pharmacologically blocked. These data provide genetic verification for the relative contributions of 6-aminopurine metabolizing pathways in L. donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation.

  5. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

    PubMed

    Hesse, Julia; Leberling, Stella; Boden, Elisabeth; Friebe, Daniela; Schmidt, Timo; Ding, Zhaoping; Dieterich, Peter; Deussen, Andreas; Roderigo, Claudia; Rose, Christine R; Floss, Doreen M; Scheller, Jürgen; Schrader, Jürgen

    2017-03-31

    Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation in addition strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

  6. Regulatory T Cells from Colon Cancer Patients Inhibit Effector T-cell Migration through an Adenosine-Dependent Mechanism.

    PubMed

    Sundström, Patrik; Stenstad, Hanna; Langenes, Veronica; Ahlmanner, Filip; Theander, Lisa; Ndah, Tapuka Gordon; Fredin, Kamilla; Börjesson, Lars; Gustavsson, Bengt; Bastid, Jérémy; Quiding-Järbrink, Marianne

    2016-03-01

    T cell-mediated immunity is a major component of antitumor immunity. In order to be efficient, effector T cells must leave the circulation and enter into the tumor tissue. Regulatory T cells (Treg) from gastric cancer patients, but not from healthy volunteers, potently inhibit migration of conventional T cells through activated endothelium. In this study, we compared T cells from colon cancer patients and healthy donors to determine the mechanisms used by Tregs from cancer patients to inhibit conventional T-cell migration. Our results showed that circulating Tregs from cancer patients expressed high levels of CD39, an ectoenzyme mediating hydrolysis of ATP to AMP, as a rate-determining first step in the generation of immunosuppressive adenosine. Tumor-associated Tregs expressed even more CD39, and we therefore examined the importance of adenosine in Treg-mediated inhibition of T-cell transendothelial migration in vitro. Exogenous adenosine significantly reduced migration of conventional T cells from healthy volunteers, and blocking either adenosine receptors or CD39 enzymatic activity during transmigration restored the ability of conventional T cells from cancer patients to migrate. Adenosine did not directly affect T cells or endothelial cells, but reduced the ability of monocytes to activate the endothelium. Taken together, our results indicate that Treg-derived adenosine acts on monocytes and contributes to reduced transendothelial migration of effector T cells into tumors. This effect of Tregs is specific for cancer patients, and our results indicate that Tregs may affect not only T-cell effector functions but also their migration into tumors.

  7. Human adenosine deaminase. Distribution and properties.

    PubMed

    Van der Weyden, M B; Kelley, W N

    1976-09-25

    Adenosine deaminase exists in multiple molecular forms in human tissue. One form of the enzyme appears to be "particulate". Three forms of the enzyme are soluble and interconvertible with apparent molecular weights of approximately 36,000, 114,000, and 298,000 (designated small, intermediate, and large, respectively). The small form of adenosine deaminase is convertible to the large form only in the presence of a protein, which has an apparent molecular weight of 200,000 and has no adenosine deaminase activity. This conversion of the small form of the enzyme to the large form occurs at 4 degrees, exhibits a pH optimum of 5.0 to 8.0, and is associated with a loss of conversion activity. The small form of the enzyme predominates in tissue preparations exhibiting the higher enzyme-specific activities and no detectable conversion activity. The large form of adenosine deaminase predominates in tissue extracts exhibiting the lower enzyme specific activities and abundant conversion activity. The small form of adenosine deaminase shows several electrophoretic variants by isoelectric focusing. The electrophoretic heterogeneity observed with the large form of the enzyme is similar to that observed with the small form, with the exception that several additional electrophoretic variants are uniformly identified. No organ specificity is demonstrable for the different electrophoretic forms. The kinetic characteristics of the three soluble molecular species of adenosine deaminase are identical except for pH optimum, which is 5.5 for the intermediate species and 7.0 to 7.4 for the large and small forms.

  8. A new class of adenosine receptors in brain: Characterization by 2-chloro( sup 3 H)adenosine binding

    SciTech Connect

    Chin, Jerome Hsicheng.

    1988-01-01

    Considerable evidence has accumulated in recent years to support a role for adenosine as an important physiological modulator in many mammalian tissues. In brain, adenosine is a potent depressant of neuronal firing and synaptic transmission. The exact mechanisms by which adenosine analogs depress nerve cell activity in the brain are not clear. Despite considerable investigation, neither the A1 nor the A2 adenosine receptors associated with adenylate cyclase have been able to account adequately for the actions of adenosine in brain. It has been proposed that additional adenosine receptors, possibly linked to calcium channels, are present in the central nervous system and are responsible for the physiological actions of adenosine. In this thesis, evidence is provided for the existence of a novel class of adenosine receptors in rat brain. The methods used to identify this new class of receptors involved radioligand binding techniques which have been successfully employed to characterize the properties of many neurotransmitter and drug receptors. 2-Chloro({sup 3}H)adenosine (Cl({sup 3}H)Ado) was selected as the ligand for these experiments since is a water-soluble, metabolically-stable analog of adenosine and a potent depressant of synaptic transmission in brain. The results demonstrate the presence of a distinct class of 2-chloro({sup 3}H)adenosine binding sites in rat forebrain membranes with an apparent K{sub D} of about 10 {mu}M and a B{sub max} of about 60 pmol per mg of protein. Specific 2-chloro ({sup 3}H)adenosine binding is highly specific for adenosine agonists and antagonists. Inhibition of binding by adenosine agonists exhibits an order of potency 2-chloroadenosine > 5{prime}-N-ethylcarboxamide adenosine > ({minus})-N{sup 6}-(R-phenylisopropyl)adenosine, which differs from that of both A1 and A2 adenosine receptors.

  9. Retinal Changes in an ATP-Induced Model of Retinal Degeneration.

    PubMed

    Aplin, Felix P; Vessey, Kirstan A; Luu, Chi D; Guymer, Robyn H; Shepherd, Robert K; Fletcher, Erica L

    2016-01-01

    In rodents and felines, intravitreal administration of adenosine triphosphate (ATP) has been shown to induce photoreceptor death providing a tractable model of retinal degeneration in these species. This study investigated the long term effects of photoreceptor loss in an ATP induced feline model of retinal degeneration. Six normal sighted felines were unilaterally blinded using intravitreal ATP injections and assessed using electroretinography (ERG) and optical coherence tomography (OCT). At 30 h (n = 3) or 12 weeks (n = 3) post-injection, the animals were euthanized and the eyes enucleated. Retinae were sectioned and labeled using immunohistochemistry for markers of cell death, neural remodeling and gliosis. Ongoing cell death and retinal degeneration was observed in the outer retina at both 30 h and 12 weeks following unilateral ATP injection. Markers of mid to late-stage retinal remodeling such as cell displacement and aberrant neurite growth were observed in the inner retina at 12 weeks post-injection. Ganglion cells appeared to remain intact in ATP injected eyes. Müller cell gliosis was observed throughout the inner and outer retina, in some parts completely enveloping and/or displacing the surviving neural tissue. Our data suggests that the ATP injected feline retina continues to undergo progressive retinal degeneration and exhibits abnormalities consistent with a description of retinal remodeling commonly seen in other models of retinal degeneration. These findings validate the use of intravitreal ATP injection in feline as a large animal model of retinal degeneration which may aid in development of therapies aiming to restore visual function after photoreceptor degeneration.

  10. Retinal Changes in an ATP-Induced Model of Retinal Degeneration

    PubMed Central

    Aplin, Felix P.; Vessey, Kirstan A.; Luu, Chi D.; Guymer, Robyn H.; Shepherd, Robert K.; Fletcher, Erica L.

    2016-01-01

    In rodents and felines, intravitreal administration of adenosine triphosphate (ATP) has been shown to induce photoreceptor death providing a tractable model of retinal degeneration in these species. This study investigated the long term effects of photoreceptor loss in an ATP induced feline model of retinal degeneration. Six normal sighted felines were unilaterally blinded using intravitreal ATP injections and assessed using electroretinography (ERG) and optical coherence tomography (OCT). At 30 h (n = 3) or 12 weeks (n = 3) post-injection, the animals were euthanized and the eyes enucleated. Retinae were sectioned and labeled using immunohistochemistry for markers of cell death, neural remodeling and gliosis. Ongoing cell death and retinal degeneration was observed in the outer retina at both 30 h and 12 weeks following unilateral ATP injection. Markers of mid to late-stage retinal remodeling such as cell displacement and aberrant neurite growth were observed in the inner retina at 12 weeks post-injection. Ganglion cells appeared to remain intact in ATP injected eyes. Müller cell gliosis was observed throughout the inner and outer retina, in some parts completely enveloping and/or displacing the surviving neural tissue. Our data suggests that the ATP injected feline retina continues to undergo progressive retinal degeneration and exhibits abnormalities consistent with a description of retinal remodeling commonly seen in other models of retinal degeneration. These findings validate the use of intravitreal ATP injection in feline as a large animal model of retinal degeneration which may aid in development of therapies aiming to restore visual function after photoreceptor degeneration. PMID:27199678

  11. Primitive ATP-activated P2X receptors: discovery, function and pharmacology

    PubMed Central

    Fountain, Samuel J.

    2013-01-01

    Adenosine 5-triphosphate (ATP) is omnipresent in biology. It is therefore no surprise that organisms have evolved multifaceted roles for ATP, exploiting its abundance and restriction of passive diffusion across biological membranes. A striking role is the emergence of ATP as a bona fide transmitter molecule, whereby the movement of ATP across membranes serves as a chemical message through a direct ligand-receptor interaction. P2X receptors are ligand-gated ion channels that mediate fast responses to the transmitter ATP in mammalian cells including central and sensory neurons, vascular smooth muscle, endothelium, and leukocytes. Molecular cloning of P2X receptors and our understanding of structure-function relationships has provided sequence information with which to query an exponentially expanding wealth of genome sequence information including protist, early animal and human pathogen genomes. P2X receptors have now been cloned and characterized from a number of simple organisms. Such work has led to surprising new cellular roles for the P2X receptors family and an unusual phylogeny, with organisms such as Drosophila and C. elegans notably lacking P2X receptors despite retaining ionotropic receptors for other common transmitters that are present in mammals. This review will summarize current work on the evolutionary biology of P2X receptors and ATP as a signaling molecule, discuss what can be drawn from such studies when considering the action of ATP in higher animals and plants, and outline how simple organisms may be exploited experimentally to inform P2X receptor function in a wider context. PMID:24367292

  12. Terbium ion-coordinated carbon dots for fluorescent aptasensing of adenosine 5'-triphosphate with unmodified gold nanoparticles.

    PubMed

    Xu, Mingdi; Gao, Zhuangqiang; Zhou, Qian; Lin, Youxiu; Lu, Minghua; Tang, Dianping

    2016-12-15

    This work reports on a novel time-resolved fluorescent aptasensing platform for the quantitative monitoring of adenosine 5'-triphosphate (ATP) by interaction of dispersive/agglomerate gold nanoparticles (AuNPs) with terbium ion-coordinated carbon dots (Tb-CDs). To construct such a fluorescent nanoprobe, Tb-CDs with high-efficient fluorescent intensity are first synthesized by the microwave method with terbium ions (Tb(3+)). The aptasensing system consists of ATP aptamer, AuNP and Tb-CD. The dispersive/agglomerate gold nanoparticles are acquired through the reaction of the aptamer with target ATP. Upon target ATP introduction, the aptamers bind with the analytes to form new aptamer-ATP complexes and coat on the surface of AuNPs to inhibit their aggregation in the high salt solution. In this case, the fluorescent signal of Tb-CDs is quenched by the dispersive AuNPs on the basis of the fluorescence resonance energy transfer (FRET). At the absence of target analyte, gold nanoparticles tend to aggregate in the high salt state even if the aptamers are present. Thus, the added Tb-CDs maintain their intrinsic fluorescent intensity. Experimental results indicated that the aptasensing system exhibited good fluorescent responses toward ATP in the dynamic range from 40nM to 4.0μM with a detection limit of 8.5nM at 3sblank criterion. The repeatability and intermediate precision is less than 9.5% at three concentrations including 0.04, 0.4 and 2.0μM ATP. The selectivity was acceptable toward guanosine 5'-triphosphate, uridine 5'-triphosphate and cytidine 5'-triphosphate. The methodology was applied to evaluate the blank human serum spiked with target ATP, and the recoveries (at 3 concentration levels) ranged between 97.0% and 103.7%. Importantly, this detection scheme is rapid, simple, cost-effective, and does not require extensive sample preparation or separation.

  13. On the Functional Role of the {epsilon} Subunit of the Molecular Motor F-Adenosine Triphosphatase in Lipid Membranes of Cells

    SciTech Connect

    Pikin, S. A. Loginov, E. B.

    2010-11-15

    The effect of the e subunit of the molecular motor F-adenosine triphosphatase, which is built into the lipid membrane of a cell, on the dynamics of the rotor ({gamma} subunit), with which this subunit is bound, has been qualitatively considered. It is shown that its structural and conformational features arising during the hydrolysis of 'fuel' adenosine triphosphate (ATP) molecules can be explained by the change in the potential within which the rotor is located. As the numerical calculations showed, at a low ATP concentration, the hydrolysis is accompanied by an unstable rotation of the {gamma} subunit and the related proton current. A model is proposed to describe the interaction between the {epsilon} subunit and the lipid order fluctuations caused by the membrane transition to the gel state. It is demonstrated that the rotor rotations become inhomogeneous when this interaction is enhanced with a decrease in the cell temperature.

  14. Regulation of adenosine transport by acute and chronic ethanol exposure

    SciTech Connect

    Nagy, L.E.; Casso, D.; Diamond, I.; Gordon, A.S. )

    1989-02-09

    Chronic exposure to ethanol results in a desensitization of adenosine receptor-stimulated cAMP production. Since adenosine is released by cells and is known to desensitize its own as well as other receptors, it may be involved in ethanol-induced desensitization of adenosine receptor function. Therefore, we have examine the acute and chronic effects of ethanol on the transport of adenosine via the nucleoside transport. Acute exposure to ethanol caused an inhibition of adenosine uptake in S49 lymphoma cells. This decrease in uptake resulted in accumulation of extracellular adenosine after ethanol exposure. The effect of ethanol was specific to nucleoside transport. Uptake of uridine, also transported by the nucleoside transporter, was inhibited by ethanol to the same degree as adenosine uptake, while neither isoleucine nor deoxyglucose uptake was altered by ethanol treatment. Inhibition of adenosine uptake by ethanol was non-competitive and dependent on the concentration of ethanol. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol. There was no longer an acute inhibition of adenosine uptake, nor was these accumulation of extracellular adenosine. Chronic ethanol exposure also resulted in a decrease in the absolute rate of adenosine uptake. Binding studies using a high affinity lignad for the nucleoside transporter, nitrobenzylthioinosine (NBMPR), indicate that this decreased uptake was due to a decrease in the maximal number of binding sites. These ethanol-induced changes in adenosine transport may be important for the acute and chronic effects of ethanol.

  15. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    PubMed

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases.

  16. Effect of exogenous adenosine and monensin on glycolytic flux in isolated perfused normoxic rat hearts: role of pyruvate kinase.

    PubMed

    Peltier, S; Burelle, Y; Novel-Chate, V; Demaison, L; Verdys, M; Saks, V; Keriel, C; Leverve, X M

    2005-09-01

    We studied the effect of exogenous adenosine in isolated perfused normoxic rat hearts on glycolytic flux through pyruvate kinase (PK). We compared its effect with that of myxothiazol, an inhibitor of mitochondrial ATP production. Moreover, we tested whether an increase of membrane ionic flux with monensin is linked to a stimulation of glycolytic flux through PK. After a 20-min stabilization period adenosine, myxothiazol or monensin were administrated to the perfusate continuously at various concentrations during 10 min. The contraction was monitored and the lactate production in coronary effluents evaluated. The amount of adenine nucleotides and phosphoenolpyruvate was measured in the frozen hearts. Myxothiazol induced a decrease of the left ventricular developed pressure (LVDP : -40%) together with a stimulation of glycolytic flux secondary to PK activation. In contrast, adenosine primarily reduced heart rate (HR: -30%) with only marginal effects on LVDP. This was associated with an inhibition of glycolysis at the level of PK. The Na+ ionophore monensin affected HR (+14%) and LVDP (+25%). This effect was associated with a stimulation of glycolysis secondary to the stimulation of PK. These results provide new information of action of adenosine in the heart and support the concept of a direct coupling between glycolysis and process regulating sarcolemmal ionic fluxes.

  17. Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells.

    PubMed

    Romio, Michael; Reinbeck, Benjamin; Bongardt, Sabine; Hüls, Sandra; Burghoff, Sandra; Schrader, Jürgen

    2011-08-01

    CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

  18. Kinetics of the acid pump in the stomach. Proton transport and hydrolysis of ATP and p-nitrophenyl phosphate by the gastric H,K-ATPase

    SciTech Connect

    Ljungstroem, M.M.; Mardh, S.

    1985-05-10

    Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity and one low affinity site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site. Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.

  19. Energy Equivalence of Information in the Mitochondrion and the Thermodynamic Efficiency of ATP Synthase.

    PubMed

    Matta, Chérif F; Massa, Lou

    2015-09-01

    Half a century ago, Johnson and Knudsen resolved the puzzle of the apparent low efficiency of the kidney (∼ 0.5%) compared to most other bodily organs (∼ 40%) by taking into account the entropic cost of ion sorting, the principal function of this organ. Similarly, it is shown that the efficiency of energy transduction of the chemiosmotic proton-motive force by ATP synthase is closer to 90% instead of the oft-quoted textbook value of only 60% when information theoretic considerations are applied to the mitochondrion. This high efficiency is consistent with the mechanical energy transduction of ATP synthase known to be close to the 100% thermodynamic limit. It would have been wasteful for evolution to maximize the mechanical energy transduction to 100% while wasting 40% of the chemiosmotic free energy in the conversion of the proton-motive force into mechanical work before being captured as chemical energy in adenosine 5'-triphosphate.

  20. Chemically inhibited ATP synthesis promoted detachment of different-age biofilms from membrane surface.

    PubMed

    Xu, Huijuan; Teo, Kaiwen; Neo, Huiling; Liu, Yu

    2012-08-01

    This study investigated the response of different-age biofilms developed on membrane surface to a chemical uncoupler 3, 3', 4', 5-tetrachlorosalicylanilide (TCS). Results showed that adenosine triphosphate (ATP) dissipation caused by TCS would promote different-age biofilms detachment, whereas chemically inhibited cellular ATP synthesis subsequently suppressed autoinducer-2 (AI-2) and extracellular polymeric substances (EPS) production. The extent of biofilm detachment was found to be closely related to AI-2-regulated EPS contents of bacteria. It was revealed that energy dissipation induced biofilm detachability was controlled by AI-2 regulated cellular communication via AI-2-mediated EPS secretion. This study would lead to a new cleaning strategy of biologically fouled membrane.

  1. Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation

    PubMed Central

    Kłosowska, Agnieszka; Chamera, Tomasz; Liberek, Krzysztof

    2016-01-01

    Hsp104 disaggregase provides thermotolerance in yeast by recovering proteins from aggregates in cooperation with the Hsp70 chaperone. Protein disaggregation involves polypeptide extraction from aggregates and its translocation through the central channel of the Hsp104 hexamer. This process relies on adenosine triphosphate (ATP) hydrolysis. Considering that Hsp104 is characterized by low affinity towards ATP and is strongly inhibited by adenosine diphosphate (ADP), we asked how Hsp104 functions at the physiological levels of adenine nucleotides. We demonstrate that physiological levels of ADP highly limit Hsp104 activity. This inhibition, however, is moderated by the Hsp70 chaperone, which allows efficient disaggregation by supporting Hsp104 binding to aggregates but not to non-aggregated, disordered protein substrates. Our results point to an additional level of Hsp104 regulation by Hsp70, which restricts the potentially toxic protein unfolding activity of Hsp104 to the disaggregation process, providing the yeast protein-recovery system with substrate specificity and efficiency in ATP consumption. DOI: http://dx.doi.org/10.7554/eLife.15159.001 PMID:27223323

  2. Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

    PubMed

    Harlan, J; DeChatelet, L R; Iverson, D B; McCall, C E

    1977-02-01

    The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.

  3. The role of adenosine in Alzheimer's disease.

    PubMed

    Rahman, Anisur

    2009-09-01

    Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system manifested by cognitive and memory deterioration, a variety of neuropsychiatric symptoms, behavioral disturbances, and progressive impairment of daily life activities. Current pharmacotherapies are restricted to symptomatic interventions but do not prevent progressive neuronal degeneration. Therefore, new therapeutic strategies are needed to intervene with these progressive pathological processes. In the past several years adenosine, a ubiquitously released purine ribonucleoside, has become important for its neuromodulating capability and its emerging positive experimental effects in neurodegenerative diseases. Recent research suggests that adenosine receptors play important roles in the modulation of cognitive function. The present paper attempts to review published reports and data from different studies showing the evidence of a relationship between adenosinergic function and AD-related cognitive deficits. Epidemiological studies have found an association between coffee (a nonselective adenosine receptor antagonist) consumption and improved cognitive function in AD patients and in the elderly. Long-term administration of caffeine in transgenic animal models showed a reduced amyloid burden in brain with better cognitive performance. Antagonists of adenosine A2A receptors mimic these beneficial effects of caffeine on cognitive function. Neuronal cell cultures with amyloid beta in the presence of an A2A receptor antagonist completely prevented amyloid beta-induced neurotoxicity. These findings suggest that the adenosinergic system constitutes a new therapeutic target for AD, and caffeine and A2A receptor antagonists may have promise to manage cognitive dysfunction in AD.

  4. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  5. Hydrolysable ATP is a requirement for the correct interaction of molecular chaperonins cpn60 and cpn10.

    PubMed Central

    Walters, Chris; Errington, Neil; Rowe, Arther J; Harding, Stephen E

    2002-01-01

    Over recent years the binding ability of the molecular chaperone cpn60 (GroEL14) and its co-chaperone cpn10 (GroES7) has been reported to occur under an assortment of specific conditions from the use of non-hydrolysable ATP analogues (namely adenosine 5'-[gamma-thio]triphosphate) to requiring hydrolysable ATP for any interaction to occur. We have investigated this further using the molecular hydrodynamic methods (hydrodynamic bead modelling, sedimentation-velocity analytical ultracentrifugation and dynamic light-scattering), allowing the process to be followed under physiologically relevant dilute solution conditions, combined with absorption spectrophotometry to determine GroES7-GroEL14 interaction through the rate inhibition of the cpn60's ATPase activity by GroES7. The results found here indicate that the presence of hydrolysable ATP is required to facilitate correct GroES7 interaction with GroEL14 in solution. PMID:12049650

  6. Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

    PubMed

    Caiazzo, Elisabetta; Tedesco, Idolo; Spagnuolo, Carmela; Russo, Gian Luigi; Ialenti, Armando; Cicala, Carla

    2016-06-01

    Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

  7. Hierarchical self-assembly of squaraine and silica nanoparticle functionalized with cationic coordination sites for near infrared detection of ATP

    PubMed Central

    Feng, Ruizhi; Shi, Weining; Wang, Dejia; Wen, Jia; Li, Hongjuan; Sun, Shiguo; Xu, Yongqian

    2017-01-01

    Optical activity of hierarchical supramolecular assemblies based on organic dyes would create multiple functional architectures. In this work, three kinds of silica nanoparticles with or without functional groups were synthesized. For the first time, silica nanoparticles can induce positively charged squaraine (SQ) to aggregate to form supramolecular assemblies. Adenosine-5′-triphosphate (ATP) as building blocks was absorbed on the surface of silica nanoparticles through metal-anion coordination and electrostatic interactions, in which the aggregates of SQ was transferred to monomer. The thickness being composed of ATP and SQ on the outside of nanoparticles is about 5 nm. These supramolecular assemblies showed selective turn-on fluorescence response to ATP in near infrared (NIR) region over other ions through metal-anion coordination and electrostatic interactions. These functional silica nanoparticles possessing many advantages provide proof-of-principle “seed crystals” for construction of supramolecular assemblies and platforms for sensing with facile performance. PMID:28240255

  8. 3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria

    NASA Astrophysics Data System (ADS)

    Renz, Anja; Renz, Marc; Klütsch, Diana; Deckers-Hebestreit, Gabriele; Börsch, Michael

    2015-03-01

    FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells.

  9. ATP-Responsive and Near-Infrared-Emissive Nanocarriers for Anticancer Drug Delivery and Real-Time Imaging

    PubMed Central

    Qian, Chenggen; Chen, Yulei; Zhu, Sha; Yu, Jicheng; Zhang, Lei; Feng, Peijian; Tang, Xin; Hu, Quanyin; Sun, Wujin; Lu, Yue; Xiao, Xuanzhong; Shen, Qun-Dong; Gu, Zhen

    2016-01-01

    Stimuli-responsive and imaging-guided drug delivery systems hold vast promise for enhancement of therapeutic efficacy. Here we report an adenosine-5'-triphosphate (ATP)-responsive and near-infrared (NIR)-emissive conjugated polymer-based nanocarrier for the controlled release of anticancer drugs and real-time imaging. We demonstrate that the conjugated polymeric nanocarriers functionalized with phenylboronic acid tags on surface as binding sites for ATP could be converted to the water-soluble conjugated polyelectrolytes in an ATP-rich environment, which promotes the disassembly of the drug carrier and subsequent release of the cargo. In vivo studies validate that this formulation exhibits promising capability for inhibition of tumor growth. We also evaluate the metabolism process by monitoring the fluorescence signal of the conjugated polymer through the in vivo NIR imaging. PMID:27217838

  10. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

    PubMed

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-09-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

  11. Inhibition of Salmonella enterica biofilm formation using small-molecule adenosine mimetics.

    PubMed

    Koopman, Jacob A; Marshall, Joanna M; Bhatiya, Aditi; Eguale, Tadesse; Kwiek, Jesse J; Gunn, John S

    2015-01-01

    Biofilms have been widely implicated in chronic infections and environmental persistence of Salmonella enterica, facilitating enhanced colonization of surfaces and increasing the ability of the bacteria to be transmitted to new hosts. Salmonella enterica serovar Typhi biofilm formation on gallstones from humans and mice enhances gallbladder colonization and bacterial shedding, while Salmonella enterica serovar Typhimurium biofilms facilitate long-term persistence in a number of environments important to food, medical, and farming industries. Salmonella regulates expression of many virulence- and biofilm-related processes using kinase-driven pathways. Kinases play pivotal roles in phosphorylation and energy transfer in cellular processes and possess an ATP-binding pocket required for their functions. Many other cellular proteins also require ATP for their activity. Here we test the hypothesis that pharmacological interference with ATP-requiring enzymes utilizing adenosine mimetic compounds would decrease or inhibit bacterial biofilm formation. Through the screening of a 3,000-member ATP mimetic library, we identified a single compound (compound 7955004) capable of significantly reducing biofilm formation by S. Typhimurium and S. Typhi. The compound was not bactericidal or bacteriostatic toward S. Typhimurium or cytotoxic to mammalian cells. An ATP-Sepharose affinity matrix technique was used to discover potential protein-binding targets of the compound and identified GroEL and DeoD. Compound 7955004 was screened against other known biofilm-forming bacterial species and was found to potently inhibit biofilms of Acinetobacter baumannii as well. The identification of a lead compound with biofilm-inhibiting capabilities toward Salmonella provides a potential new avenue of therapeutic intervention against Salmonella biofilm formation, with applicability to biofilms of other bacterial pathogens.

  12. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats

    PubMed Central

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-01-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc. PMID:24639102

  13. A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    PubMed Central

    Jones, Karlie R.; Choi, Uimook; Gao, Ji-Liang; Thompson, Robert D.; Rodman, Larry E.; Malech, Harry L.; Kang, Elizabeth M.

    2017-01-01

    Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds. PMID:28317879

  14. Adenosine triphosphate prevents serum deprivation-induced apoptosis in human mesenchymal stem cells via activation of the MAPK signaling pathways.

    PubMed

    Berlier, Jessica L; Rigutto, Sabrina; Dalla Valle, Antoine; Lechanteur, Jessica; Soyfoo, Muhammad S; Gangji, Valerie; Rasschaert, Joanne

    2015-01-01

    Human mesenchymal stem cells (hMSC) are multipotent cells derived from various sources including adipose and placental tissues as well as bone marrow. Owing to their regenerative and immunomodulatory properties, their use as a potential therapeutic tool is being extensively tested. However, one of the major hurdles in using cell-based therapy is the use of fetal bovine serum that can trigger immune responses, viral and prion diseases. The development of a culture medium devoid of serum while preserving cell viability is therefore a major challenge. In this study, we demonstrated that adenosine triphosphate (ATP) restrained serum deprivation-induced cell death in hMSC by preventing caspases 3/7 activation and modulating ERK1/2 and p38 MAPK signaling pathways. We also showed that serum deprivation conditions triggered dephosphorylation of the proapoptotic protein Bad leading to cell death. Adjunction of ATP restored the phosphorylation state of Bad. Furthermore, ATP significantly modulated the expression of proapoptopic and antiapoptotic genes, in favor of an antiapoptotic profile expression. Finally, we established that hMSC released a high amount of ATP in the extracellular medium when cultured in a serum-free medium. Collectively, our results demonstrate that ATP favors hMSC viability in serum deprivation conditions. Moreover, they shed light on the cardinal role of the MAPK pathways, ERK1/2 and p38 MAPK, in promoting hMSC survival.

  15. Adenosine receptors and the central nervous system.

    PubMed

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    The adenosine receptors (ARs) in the nervous system act as a kind of "go-between" to regulate the release of neurotransmitters (this includes all known neurotransmitters) and the action of neuromodulators (e.g., neuropeptides, neurotrophic factors). Receptor-receptor interactions and AR-transporter interplay occur as part of the adenosine's attempt to control synaptic transmission. A(2A)ARs are more abundant in the striatum and A(1)ARs in the hippocampus, but both receptors interfere with the efficiency and plasticity-regulated synaptic transmission in most brain areas. The omnipresence of adenosine and A(2A) and A(1) ARs in all nervous system cells (neurons and glia), together with the intensive release of adenosine following insults, makes adenosine a kind of "maestro" of the tripartite synapse in the homeostatic coordination of the brain function. Under physiological conditions, both A(2A) and A(1) ARs play an important role in sleep and arousal, cognition, memory and learning, whereas under pathological conditions (e.g., Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, epilepsy, drug addiction, pain, schizophrenia, depression), ARs operate a time/circumstance window where in some circumstances A(1)AR agonists may predominate as early neuroprotectors, and in other circumstances A(2A)AR antagonists may alter the outcomes of some of the pathological deficiencies. In some circumstances, and depending on the therapeutic window, the use of A(2A)AR agonists may be initially beneficial; however, at later time points, the use of A(2A)AR antagonists proved beneficial in several pathologies. Since selective ligands for A(1) and A(2A) ARs are now entering clinical trials, the time has come to determine the role of these receptors in neurological and psychiatric diseases and identify therapies that will alter the outcomes of these diseases, therefore providing a hopeful future for the patients who suffer from these diseases.

  16. ATP contributes to the generation of network-driven giant depolarizing potentials in the neonatal rat hippocampus

    PubMed Central

    Safiulina, Victoria F; Kasyanov, Alexander M; Sokolova, Elena; Cherubini, Enrico; Giniatullin, Rashid

    2005-01-01

    In the immature hippocampus, the so-called ‘giant depolarizing potentials’ (GDPs) are network-driven synaptic events generated by the synergistic action of glutamate and GABA. Here we tested the hypothesis that ATP, a widely distributed neurotransmitter, directly contributes to the network activity during the first postnatal week. We found that in CA3 pyramidal cells, in the presence of the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), ATP produced a transient facilitation of GDPs followed by a depressant effect. A similar biphasic effect was produced by blockade of the ectoATPase activity with 6-N,N-diethyl-d-β,γ-dibromomethylene ATP (ARL-67156). The effects of exogenous and endogenous ATP on GDPs were prevented by the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). On pyramidal cells, ATP upregulated spontaneous action-potential-dependent GABAA-mediated synaptic events (GABA-SPSPs), suggesting a network-driven effect. Recordings from interneurones allowed comparison of ATP effects on GABAergic and glutamatergic synaptic activity. While ATP depressed GABA-SPSPs via metabotropic P2Y1 receptors, it up- and downregulated glutamatergic SPSPs via PPADS-sensitive receptors. Thus, ATP exerts an excitatory action on CA3 pyramidal cells via facilitation of GDPs and SPSPs. This excitatory drive is propagated to pyramidal cells by interneurons that represent the ‘common pathway’ for generation of GDPs and SPSPs. Our results show that ATP operating via distinct P2X and P2Y receptors directly contributes to modulate network activity at the early stages of postnatal development. PMID:15845583

  17. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline.

    PubMed

    Preiss, Laura; Langer, Julian D; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E G; Koul, Anil; Meier, Thomas

    2015-05-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring's ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens.

  18. Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline

    PubMed Central

    Preiss, Laura; Langer, Julian D.; Yildiz, Özkan; Eckhardt-Strelau, Luise; Guillemont, Jérôme E. G.; Koul, Anil; Meier, Thomas

    2015-01-01

    Multidrug-resistant tuberculosis (MDR-TB) is more prevalent today than at any other time in human history. Bedaquiline (BDQ), a novel Mycobacterium-specific adenosine triphosphate (ATP) synthase inhibitor, is the first drug in the last 40 years to be approved for the treatment of MDR-TB. This bactericidal compound targets the membrane-embedded rotor (c-ring) of the mycobacterial ATP synthase, a key metabolic enzyme required for ATP generation. We report the x-ray crystal structures of a mycobacterial c9 ring without and with BDQ bound at 1.55- and 1.7-Å resolution, respectively. The structures and supporting functional assays reveal how BDQ specifically interacts with the rotor ring via numerous interactions and thereby completely covers the c-ring’s ion-binding sites. This prevents the rotor ring from acting as an ion shuttle and stalls ATP synthase operation. The structures explain how diarylquinoline chemicals specifically inhibit the mycobacterial ATP synthase and thus enable structure-based drug design of next-generation ATP synthase inhibitors against Mycobacterium tuberculosis and other bacterial pathogens. PMID:26601184

  19. ATP-Induced Inflammasome Activation and Pyroptosis Is Regulated by AMP-Activated Protein Kinase in Macrophages

    PubMed Central

    Zha, Qing-Bing; Wei, Hong-Xia; Li, Chen-Guang; Liang, Yi-Dan; Xu, Li-Hui; Bai, Wen-Jing; Pan, Hao; He, Xian-Hui; Ouyang, Dong-Yun

    2016-01-01

    Adenosine triphosphate (ATP) is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK) activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL)-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small-interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist). Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation. PMID:28018360

  20. Effects of adenosine infusion into renal interstitium on renal hemodynamics

    SciTech Connect

    Pawlowska, D.; Granger, J.P.; Knox, F.G.

    1987-04-01

    This study was designed to investigate the hemodynamic effects of exogenous adenosine in the interstitium of the rat kidney. Adenosine or its analogues were infused into the renal interstitium by means of chronically implanted capsules. In fusion of adenosine decreased glomerular filtration rate (GFR) from 0.81 +/- 0.06 to 0.37 +/- 0.06 ml/min while having no effect on renal blood flow (RBF). The metabolically stable analogue, 2-chloradenosine (2-ClAdo), decreased GFR from 0.73 +/- 0.07 to 021 +/- 0.06 ml/min. Interstitial infusion of theophylline, an adenosine receptor antagonist, completely abolished the effects of adenosine and 2-ClAdo on GFR. The distribution of adenosine, when infused into the renal interstitium, was determined using radiolabeled 5'-(N-ethyl)-carboxamidoadenosine (NECA), a metabolically stable adenosine agonist. After continuous infusion, (/sup 3/H)NECA was distributed throughout the kidney. The effects of NECA to reduce GFR were similar to those of adenosine and 2-ClAdo. They conclude that increased levels of adenosine in the renal interstitium markedly decrease GFR without affecting RBF in steady-state conditions. The marked effects of adenosine agonists during their infusion into the renal interstitium and the complete blockade of these effects by theophylline suggest an extracellular action of adenosine.

  1. Neuroprotective effects of adenosine deaminase in the striatum

    PubMed Central

    Tamura, Risa; Satoh, Yasushi; Nonoyama, Shigeaki; Nishida, Yasuhiro; Nibuya, Masashi

    2016-01-01

    Adenosine deaminase (ADA) is a ubiquitous enzyme that catabolizes adenosine and deoxyadenosine. During cerebral ischemia, extracellular adenosine levels increase acutely and adenosine deaminase catabolizes the increased levels of adenosine. Since adenosine is a known neuroprotective agent, adenosine deaminase was thought to have a negative effect during ischemia. In this study, however, we demonstrate that adenosine deaminase has substantial neuroprotective effects in the striatum, which is especially vulnerable during cerebral ischemia. We used temporary oxygen/glucose deprivation (OGD) to simulate ischemia in rat corticostriatal brain slices. We used field potentials as the primary measure of neuronal damage. For stable and efficient electrophysiological assessment, we used transgenic rats expressing channelrhodopsin-2, which depolarizes neurons in response to blue light. Time courses of electrically evoked striatal field potential (eFP) and optogenetically evoked striatal field potential (optFP) were recorded during and after oxygen/glucose deprivation. The levels of both eFP and optFP decreased after 10 min of oxygen/glucose deprivation. Bath-application of 10 µg/ml adenosine deaminase during oxygen/glucose deprivation significantly attenuated the oxygen/glucose deprivation-induced reduction in levels of eFP and optFP. The number of injured cells decreased significantly, and western blot analysis indicated a significant decrease of autophagic signaling in the adenosine deaminase-treated oxygen/glucose deprivation slices. These results indicate that adenosine deaminase has protective effects in the striatum. PMID:26746865

  2. On the use of X-ray absorption spectroscopy to elucidate the structure of lutetium adenosine mono- and triphosphate complexes.

    PubMed

    Mostapha, S; Berthon, C; Fontaine-Vive, F; Gaysinski, M; Guérin, L; Guillaumont, D; Massi, L; Monfardini, I; Solari, P L; Thomas, O P; Charbonnel, M C; Den Auwer, C

    2014-02-01

    Although the physiological impact of the actinide elements as nuclear toxicants has been widely investigated for half a century, a description of their interactions with biological molecules remains limited. It is however of primary importance to better assess the determinants of actinide speciation in cells and more generally in living organisms to unravel the molecular processes underlying actinide transport and deposition in tissues. The biological pathways of this family of elements in case of accidental contamination or chronic natural exposure (in the case of uranium rich soils for instance) are therefore a crucial issue of public health and of societal impact. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, phosphate derivatives are considered as probable targets of these cations. Among them, nucleotides and in particular adenosine mono- (AMP) and triphosphate (ATP) nucleotides occur in more chemical reactions than any other compounds on the earth's surface, except water, and are therefore critical target molecules. In the present study, we are interested in trans-plutonium actinide elements, in particular americium and curium that are more rarely considered in environmental and bioaccumulation studies than early actinides like uranium, neptunium and plutonium. A first step in this strategy is to work with chemical analogues like lanthanides that are not radioactive and therefore allow extended physical chemical characterization to be conducted that are difficult to perform with radioactive materials. We describe herein the interaction of lutetium(III) with adenosine AMP and ATP. With AMP and ATP, insoluble amorphous compounds have been obtained with molar ratios of 1:2 and 1:1, respectively. With an excess of ATP, with 1:2 molar ratio, a soluble complex has been obtained. A combination of spectroscopic techniques (IR, NMR, ESI-MS, EXAFS) together with quantum

  3. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces

    PubMed Central

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-01-01

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate “cleanliness” using a sampling area–adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm2, 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm2) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm2, which corresponded to culture-assay levels of <2.5 CFU/cm2. An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate. PMID:27294944

  4. Interaction of 5'-P-sulfonylbenzoyl adenosine with cysteine residues of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

    SciTech Connect

    El-Maghrabi, M.R.; Lively, M.O.; Pilkis, S.J.

    1987-05-01

    The kinase and bisphosphatase reactions of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase appear to be catalyzed at separate active sites. The kinase site contains 3 cysteinyl residues that are important for sugar phosphate binding but not for ATP binding. These groups are readily alkylated with iodoacetamide which decreases by 15-fold the affinity for Fru 6-P but also increases the maximal velocity of the reaction by the same extent. Incubation of the enzyme with 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP analog, has no effect on the bisphosphatase activity but inactivates the kinase. The addition of dithiothreitol completely reactivates the kinase, suggesting that the reagent affected sulfhydryl groups critical for sugar phosphate binding and not the ATP site of the enzyme. Similarly, 8-Azido-ATP/UV-photoinactivated enzyme is also reactivated by dithiothreitol and involves the same sulfhydryl groups, since alkylation of the latter with iodoacetamide protects the enzyme from inactivation by FSBA and from 8-azido ATP. Cyanogen bromide cleavage of enzyme that had been alkylated with iodo(I-/sup 14/C)acetamide yielded a 20,000 dalton peptide which contained the three cysteinyl residues. It is concluded that the site of action of ATP analogs to inactivate the kinase are these cysteinyl residues rather than the ATP binding site per se.

  5. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces.

    PubMed

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-06-09

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate "cleanliness" using a sampling area-adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm², 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm²) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm², which corresponded to culture-assay levels of <2.5 CFU/cm². An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate.

  6. A polycystin-type transient receptor potential (Trp) channel that is activated by ATP

    PubMed Central

    Traynor, David

    2017-01-01

    ABSTRACT ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP. PMID:28011630

  7. Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

    PubMed

    Jiang, Bo; Liu, Yu

    2013-06-01

    Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules.

  8. Anaerobic homolactate fermentation with Saccharomyces cerevisiae results in depletion of ATP and impaired metabolic activity.

    PubMed

    Abbott, Derek A; van den Brink, Joost; Minneboo, Inge M K; Pronk, Jack T; van Maris, Antonius J A

    2009-05-01

    Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae. ATP-dependent export of the lactate anion and/or proton, resulting in net zero ATP formation, is suspected as the underlying cause. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S. cerevisiae, aerobic carbon-limited chemostats were performed and subjected to anaerobic perturbations in the presence of high glucose concentrations. Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol. Finally, the anticipated increase in maximal velocity (V(max)) of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.

  9. The effect of asanguinous cardioplegic arrest on atrial preservation using atrial ATP as a marker.

    PubMed

    Hines, G L; Scheaffer, P; Williams, L; Mantell, P; Cheifitz, P

    1990-01-01

    Changes in atrial adenosine triphosphate (ATP) and the presence of postoperative arrhythmias were studied in 14 patients during routine coronary artery bypass grafting to 1) attempt to evaluate atrial preservation, and 2) determine if a relationship exists between changes in ATP and the development of postoperative arrhythmias. Atrial biopsies were obtained at the time of cannulation (preischemic sample) and after the removal of the aortic crossclamp (postischemic sample). Methods of myocardial protection included systemic hypothermia (28 degrees C), periodic reinfusion of crystalloid cardioplegia into the aortic root and completed vein grafts, and iced slush in the pericardial well. Atrial temperature was monitored. Preischemic ATP was 0.412 +/- 0.32 mu mol/gm, and the postischemic value was 0.220 +/- 0.13 mu mol/gm (p less than .02). Atrial temperature routinely decreased to 13-18 degrees C after cardioplegic infusion but rose to 24 degrees C between infusions. There was no correlation between postoperative supraventricular arrhythmias (4 patients) and changes in ATP. In conclusion, routine coronary artery bypass grafting with standard methods of cardiac preservation does not appear to satisfactorily preserve atrial tissue. The clinical correlation and significance of this remains to be elucidated.

  10. Homogeneously ultrasensitive electrochemical detection of adenosine triphosphate based on multiple signal amplification strategy.

    PubMed

    Chen, Xiaojun; Ge, Lingna; Guo, Buhua; Yan, Ming; Hao, Ning; Xu, Lin

    2014-08-15

    An ultrasensitive electrochemical aptasensor was successfully fabricated for the detection of adenosine triphosphate (ATP). For the first time, one detection system combined several elements: magnetic aptamer sequences for target recognition and separation, a DNAzyme assisted cyclic signal amplification strategy, layer-by-layer (LBL) quantum dots (QDs) composites for promoting square wave anodic stripping voltammetric (SWASV) analysis and Bi, Nafion (Nf) and three-dimensional ordered macroporous polyaniline-ionic liquid (Bi/Nf/3DOM PANI-IL) film modified glassy carbon electrode (GCE) for monitoring enhanced SWASV signal. The modification of Nf/3DOM PANI-IL on GCE showed that the preconcentration efficiency was improved by the electrostatic absorption of Cd(2+) with negative Nf layer with the enhanced analytical sensitivity due to a large active surface area of 3DOM structure. The increased SWASV peak current values of the label (CdS)4@SiO2 composites were found to be proportional to the logarithmic value of ATP concentrations in the range of 1pM-10nM and 10nM-1µM, with the detection limit as low as 0.5pM. The proposed aptasensor has shown an excellent performance such as high sensitivity, good selectivity and analytical application in real samples. The results demonstrated that the multiple signal amplified strategy we developed was feasible for clinical ATP assay and would provide a promising model for the detection of other small molecules.

  11. Aptamer-based electrochemical biosensor for detection of adenosine triphosphate using a nanoporous gold platform.

    PubMed

    Kashefi-Kheyrabadi, Leila; Mehrgardi, Masoud A

    2013-12-01

    In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4 °C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.

  12. Using silicon nanowire devices to detect adenosine triphosphate liberated from electrically stimulated HeLa cells.

    PubMed

    Chen, C C; Chen, Y-Z; Huang, Y-J; Sheu, J-T

    2011-01-15

    In this study, we used a biosensor chip featuring Abl tyrosine kinase-modified silicon nanowire field-effect transistors (SiNW-FETs) to detect adenosine triphosphate (ATP) liberated from HeLa cells that had been electrically stimulated. Cells that are cultured in high-ionic-strength media or buffer environments usually undermine the sensitivity and selectively of SiNW-FET-based sensors. Therefore, we first examined the performance of the biosensor chip incorporating the SiNW-FETs in both low- and high-ionic-strength buffer solutions. Next, we stimulated, using a sinusoidal wave (1.0 V, 50 Hz, 10 min), HeLa cells that had been cultured on a cell-culture chip featuring interdigitated electrodes. The extracellular ATP concentration increased by ca. 18.4-fold after electrical stimulation. Finally, we detected the presence of extracellular ATP after removing a small amount of buffer solution from the cell-cultured chip and introducing it into the biosensor chip.

  13. Bond cleavages of adenosine 5'-triphosphate induced by monochromatic soft X-rays

    NASA Astrophysics Data System (ADS)

    Fujii, K.; Narita, A.; Yokoya, A.

    2014-04-01

    To investigate which type of bond is likely to be cleaved by soft X-ray exposure to an adenosine 5'-triphosphate (ATP), we observed spectral changes in X-ray absorption near edge structure (XANES) around nitrogen and oxygen K-edge of an ATP film by soft X-ray irradiation. Experiments were performed at a synchrotron soft X-ray beamline at SPring-8, Japan. The XANES spectra around the nitrogen and oxygen .K-edge slightly varied by exposure to 560 eV soft X-rays. These changes are originated from the cleavage of C-N bonds between a sugar and a nucleobase site and of C-O, P-O or O-H bond of sugar and phosphate site. From the comparison between the change in XANES intensity of σ* peak at nitrogen and that at oxygen K-edges, it is inferred that the C-O, P-O or O-H bond of sugar and phosphate is much efficiently cleaved than the C-N of N-glycoside bond by the exposure of 560 eV soft X-ray to ATP film.

  14. A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

    PubMed

    Kucherenko, I S; Kucherenko, D Yu; Soldatkin, O O; Lagarde, F; Dzyadevych, S V; Soldatkin, A P

    2016-04-01

    The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples.

  15. Adenosine triphosphate bioluminescence analysis for rapid screening of microbial contamination in non-sterile pharmaceutical samples.

    PubMed

    Jimenez, Luis

    2004-01-01

    An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceutical formulations such as creams, tablets, and capsules. Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else. Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Aspergillus niger. Samples were incubated for 24-120 h at 35 degrees C with shaking. Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay. However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was confirmed. After incubation, all microorganisms were detected by the bioluminescence system within 24-72 h. All positive samples were confirmed by using standard plating media. However, to optimize detection of all microorganisms, different enrichment media were developed.

  16. Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

    PubMed

    Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.

  17. Persistence of biomarker ATP and ATP-generating capability in bacterial cells and spores contaminating spacecraft materials under earth conditions and in a simulated martian environment.

    PubMed

    Fajardo-Cavazos, Patricia; Schuerger, Andrew C; Nicholson, Wayne L

    2008-08-01

    Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5'-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m(-2) of UV-C (200 to 280 nm), -10 degrees C, and a Mars gas mix of CO(2) (95.54%), N(2) (2.7%), Ar (1.6%), O(2) (0.13%), and H(2)O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.

  18. Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment▿

    PubMed Central

    Fajardo-Cavazos, Patricia; Schuerger, Andrew C.; Nicholson, Wayne L.

    2008-01-01

    Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m−2 of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces. PMID:18567687

  19. Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis.

    PubMed

    Meurer, Florian; Do, Hoang Tam; Sadowski, Gabriele; Held, Christoph

    2017-04-01

    ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH7 and pH8 at 298.15K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of -13.72±0.75kJ·mol(-1). Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be -31.55±1.27kJ·mol(-1). For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH7, 298.15K and standard state.

  20. The aptamer DNA-templated fluorescence silver nanoclusters: ATP detection and preliminary mechanism investigation.

    PubMed

    Xu, Jiaona; Wei, Chunying

    2017-01-15

    Two general and reliable fluorescence sensors were proposed in this work utilizing aptamer DNA-templated silver nanoclusters (Ag NCs). Both DNA-AgNCs could be used for label-free detecting of ATP with the limits of detection of 0.44 and 0.65mM. One of them was further applied to monitor the activity of adenosine deaminase (ADA). In our effort to elucidate the light-up mechanism, we studied a total of six Ag NCs prepared by different DNA sequences, and found that they showed different sensitivity to ATP. Both BT3T3- and BT3T3(R)-templated Ag NCs were chose to make particular studies by UV-vis, TEM, fluorescence, and TCSPC methods. The results showed that when DNA-Ag NCs was kept for 1.5h and presented a strong fluorescence, the addition of ATP failed to cause a large change of fluorescence intensity; on the contrary, after Ag NCs was kept for 24h and emitted a weak fluorescence, adding ATP was able to result in the large fluorescence enhanced of 43 and 33 times for BT3T3- and BT3T3(R)-templated Ag NCs, respectively. The possible mechanism was also suggested that ATP binding to aptamer segment of template induced the change of the DNA secondary structure, which made the aggregated Ag nanoparticles disperse into Ag NCs with an average diameter of about 2nm that were responsible for the large fluorescence increase. Moreover, ATP could protect the fluorescence intensity of BT3T3(R)-templated Ag NCs from quenching for at least 9h.

  1. Cooperation of Adenosine with Macrophage Toll-4 Receptor Agonists Leads to Increased Glycolytic Flux through the Enhanced Expression of PFKFB3 Gene*

    PubMed Central

    Ruiz-García, Almudena; Monsalve, Eva; Novellasdemunt, Laura; Navarro-Sabaté, Àurea; Manzano, Anna; Rivero, Samuel; Castrillo, Antonio; Casado, Marta; Laborda, Jorge; Bartrons, Ramón; Díaz-Guerra, María José M.

    2011-01-01

    Macrophages activated through Toll receptor triggering increase the expression of the A2A and A2B adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A2A and A2B receptors, we demonstrate that the A2A receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A2B receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A2 receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages. PMID:21464136

  2. Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function

    PubMed Central

    Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Sinha Ray, Sougata; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

    2015-01-01

    Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts. PMID:25811190

  3. Purinergic signaling in early inflammatory events of the foreign body response: modulating extracellular ATP as an enabling technology for engineered implants and tissues.

    PubMed

    Rhett, J Matthew; Fann, Stephen A; Yost, Michael J

    2014-10-01

    Purinergic signaling is a ubiquitous and vital aspect of mammalian biology in which purines--mainly adenosine triphosphate (ATP)--are released from cells through loss of membrane integrity (cell death), exocytosis, or transport/diffusion across membrane channels, and exert paracrine or autocrine signaling effects through three subclasses of well-characterized receptors: the P1 adenosine receptors, the P2X ionotropic nucleotide receptors, and the P2Y metabotropic receptors. ATP and its metabolites are released by damaged and stressed cells in injured tissues. The early events of wound healing, hemostasis, and inflammation are highly regulated by these signals through activation of purinergic receptors on platelets and neutrophils. Recent data have demonstrated that ATP signaling is of particular importance to targeting leukocytes to sites of injury. This is particularly relevant to the subject of implanted medical devices, engineered tissues, and grafts as all these technologies elicit a wound healing response with varying degrees of encapsulation, rejection, extrusion, or destruction of the tissue or device. Here, we review the biology of purinergic signaling and focus on ATP release and response mechanisms that pertain to the early inflammatory phase of wound healing. Finally, therapeutic options are explored, including a new class of peptidomimetic drugs based on the ATP-conductive channel connexin43.

  4. Adenosine thallium 201 myocardial perfusion scintigraphy

    SciTech Connect

    Verani, M.S. )

    1991-07-01

    Pharmacologic coronary vasodilation as an adjunct to myocardial perfusion imaging has become increasingly important in the evaluation of patients with coronary artery disease, in view of the large number of patients who cannot perform an adequate exercise test or in whom contraindications render exercise inappropriate. Adenosine is a very potent coronary vasodilator and when combined with thallium 201 scintigraphy produces images of high quality, with the added advantages of a very short half-life (less than 10 seconds) and the ability to adjust the dose during the infusion, which may enhance safety and curtail the duration of side effects. The reported sensitivity and specificity of adenosine thallium 201 scintigraphy for the detection of coronary artery disease are high and at least comparable with imaging after exercise or dipyridamole administration. 23 refs.

  5. Therapeutic epilepsy research: from pharmacological rationale to focal adenosine augmentation

    PubMed Central

    Boison, Detlev; Stewart, Kerry-Ann

    2009-01-01

    Epilepsy is a common seizure disorder affecting approximately 70 million people worldwide. Current pharmacotherapy is neuron-centered, frequently accompanied by intolerable side-effects, and fails to be effective in about one third of patients. Therefore, new therapeutic concepts are needed. Recent research suggests an astrocytic basis of epilepsy, presenting the possibility of novel therapeutic targets. In particular, dysfunction of the astrocyte-controlled, endogenous, adenosine-based seizure control system of the brain is implicated in seizure generation. Thus, astrogliosis – a pathological hallmark of the epileptic brain – is associated with upregulation of the adenosine-removing enzyme adenosine kinase (ADK), resulting in focal adenosine deficiency. Both astrogliotic upregulation of ADK in epilepsy and transgenic overexpression of ADK are associated with seizures, and inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy. These findings link adenosine deficiency with seizures and predict that adenosine augmentation therapies (AATs) will likely be effective in preventing seizures. Given the widespread systemic and central side effects of systemically administered AATs, focal AATs (i.e., limited to the astrogliotic lesion) are a necessity. This Commentary will discuss the pharmacological rationale for the development of focal AATs. Additionally, several AAT strategies will be discussed: (1) adenosine released from silk-based brain implants; (2) adenosine released from locally implanted encapsulated cells; (3) adenosine released from stem cell-derived brain implants; and (4) adenosine augmenting gene therapies. Finally, new developments and therapeutic challenges in using focal AATs for epilepsy therapy will critically be evaluated. PMID:19682439

  6. Human K(ATP) channelopathies: diseases of metabolic homeostasis.

    PubMed

    Olson, Timothy M; Terzic, Andre

    2010-07-01

    Assembly of an inward rectifier K+ channel pore (Kir6.1/Kir6.2) and an adenosine triphosphate (ATP)-binding regulatory subunit (SUR1/SUR2A/SUR2B) forms ATP-sensitive K+ (KATP) channel heteromultimers, widely distributed in metabolically active tissues throughout the body. KATP channels are metabolism-gated biosensors functioning as molecular rheostats that adjust membrane potential-dependent functions to match cellular energetic demands. Vital in the adaptive response to (patho)physiological stress, KATP channels serve a homeostatic role ranging from glucose regulation to cardioprotection. Accordingly, genetic variation in KATP channel subunits has been linked to the etiology of life-threatening human diseases. In particular, pathogenic mutations in KATP channels have been identified in insulin secretion disorders, namely, congenital hyperinsulinism and neonatal diabetes. Moreover, KATP channel defects underlie the triad of developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). KATP channelopathies implicated in patients with mechanical and/or electrical heart disease include dilated cardiomyopathy (with ventricular arrhythmia; CMD1O) and adrenergic atrial fibrillation. A common Kir6.2 E23K polymorphism has been associated with late-onset diabetes and as a risk factor for maladaptive cardiac remodeling in the community-at-large and abnormal cardiopulmonary exercise stress performance in patients with heart failure. The overall mutation frequency within KATP channel genes and the spectrum of genotype-phenotype relationships remain to be established, while predicting consequences of a deficit in channel function is becoming increasingly feasible through systems biology approaches. Thus, advances in molecular medicine in the emerging field of human KATP channelopathies offer new opportunities for targeted individualized screening, early diagnosis, and tailored therapy.

  7. Nano-nutrition of chicken embryos. The effect of silver nanoparticles and ATP on expression of chosen genes involved in myogenesis.

    PubMed

    Sawosz, Filip; Pineda, Lane; Hotowy, Anna; Jaworski, Sławomir; Prasek, Marta; Sawosz, Ewa; Chwalibog, André

    2013-01-01

    It has been suggested that the quantity and quality of nutrients stored in the egg might not be optimal for the fast rate of chicken embryo development in modern broilers, and embryos could be supplemented with nutrients by in ovo injection. Recent experiments showed that in ovo feeding reduces post-hatch mortality and skeletal disorders and increases muscle growth and breast meat yield. Adenosine triphosphate (ATP) is a "ready for use" energetic molecule, while nanoparticles of silver (Nano-Ag) may penetrate tissues as well as cells and localise inside cells. In this investigation, we hypothesised that silver nanoparticles could be used as a protective carrier for ATP as well as an active agent. ATP and/or an ATP complex with Nano-Ag would be delivered to the muscle cells as a gene expression regulator and promoter of growth and development of embryo breast muscle. A collection of 160 broiler eggs was randomly divided into a Control group without injection and injected groups with hydrocolloids of Nano-Ag, ATP or a complex of Nano-Ag and ATP (Nano-Ag/ATP). The embryos were evaluated on day 20 of incubation. The results indicate that the application of ATP to chicken embryos increases expression of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and Na(+)/K(+) transporting ATPase (ATP1A1), which may indicate that an extra energy source can enhance molecular mechanisms of muscle cell proliferation. Nano-Ag also up-regulated expression of FGF2, VEGF, ATP1A1 and, also up-regulated expression of myogenic differentiation 1(MyoD1), affecting cell differentiation. The results indicate that ATP and Nano-Ag may accelerate growth and maturation of muscle cells.

  8. Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

    PubMed

    Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx.

  9. Role of adenosine in oligodendrocyte precursor maturation

    PubMed Central

    Coppi, Elisabetta; Cellai, Lucrezia; Maraula, Giovanna; Dettori, Ilaria; Melani, Alessia; Pugliese, Anna Maria; Pedata, Felicita

    2015-01-01

    Differentiation and maturation of oligodendroglial cells are postnatal processes that involve specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognized. As evaluated on OPC cultures, adenosine, by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary, by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K+ currents (IK) that are involved in the regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma. PMID:25964740

  10. ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes.

    PubMed

    Gwanyanya, Asfree; Sipido, Karin R; Vereecke, Johan; Mubagwa, Kanigula

    2006-10-01

    The Mg(2+)-inhibited cation (MIC) current (I(MIC)) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg(2+) homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I(MIC), studied in the presence or absence of extracellular divalent cations, was sustained for >or=50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I(MIC) exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I(MIC) rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 microM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by prevention of its resynthesis (10 microM wortmannin or 15 microM phenylarsine oxide) induced rundown of I(MIC). Finally, loading ATP-depleted cells with exogenous PIP(2) (10 microM) prevented rundown. These results suggest that PIP(2), likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity.

  11. Effects of adenosine perfusion on the metabolism and contractile activity of Rana ridibunda heart.

    PubMed

    Lazou, A; Beis, I

    1987-01-01

    The effects of adenosine were examined on the isolated perfused heart of the frog Rana ridibunda. Adenosine produced negative chronotropic and inotropic effects on frog ventricle in a concentration-dependent manner. The effects of adenosine on cardiac metabolism were also investigated by measuring the tissue content of adenine nucleotides, lactate, pyruvate, adenosine and inorganic phosphate, during adenosine perfusion. Adenosine had no effect on the tissue content of metabolites. No net synthesis of adenine nucleotides was observed during perfusion with increasing concentrations of adenosine. Lactate output from the heart decreased significantly with adenosine perfusion. Correlation of adenosine effects on cardiac muscle with the effects of hypoxia are discussed.

  12. High-Throughput Screening for RecA Inhibitors Using a Transcreener Adenosine 5′-O-Diphosphate Assay

    PubMed Central

    Peterson, Eliza J.R.; Janzen, William P.; Kireev, Dmitri

    2012-01-01

    Abstract The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5′-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener® adenosine 5′-O-diphosphate [ADP]2 fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z′=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC50 values ≤10 μM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays. PMID:22192312

  13. High-throughput screening for RecA inhibitors using a transcreener adenosine 5'-O-diphosphate assay.

    PubMed

    Peterson, Eliza J R; Janzen, William P; Kireev, Dmitri; Singleton, Scott F

    2012-06-01

    The activities of the bacterial RecA protein are involved in the de novo development and transmission of antibiotic resistance genes, thus allowing bacteria to overcome the metabolic stress induced by antibacterial agents. RecA is ubiquitous and highly conserved among bacteria, but has only distant homologs in human cells. Together, this evidence points to RecA as a novel and attractive antibacterial drug target. All known RecA functions require the formation of a complex formed by multiple adenosine 5'-O-triphosphate (ATP)-bound RecA monomers on single-stranded DNA. In this complex, RecA hydrolyzes ATP. Although several methods for assessing RecA's ATPase activity have been reported, these assay conditions included relatively high concentrations of enzyme and ATP and thereby restricted the RecA conformational state. Herein, we describe the validation of commercial reagents (Transcreener(®) adenosine 5'-O-diphosphate [ADP](2) fluorescence polarization assay) for the high-throughput measurement of RecA's ATPase activity with lower concentrations of ATP and RecA. Under optimized conditions, ADP detection by the Transcreener reagent provided robust and reproducible activity data (Z'=0.92). Using the Transcreener assay, we screened 113,477 small molecules against purified RecA protein. In total, 177 small molecules were identified as confirmed hits, of which 79 were characterized by IC(50) values ≤ 10 μM and 35 were active in bioassays with live bacteria. This set of compounds comprises previously unidentified scaffolds for RecA inhibition and represents tractable hit structures for efforts aimed at tuning RecA inhibitory activity in both biochemical and bacteriological assays.

  14. Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

    PubMed Central

    Murali, N; Lin, Y; Mechulam, Y; Plateau, P; Rao, B D

    1997-01-01

    The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain. Images FIGURE 6 PMID:9129831

  15. Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

    PubMed

    Gicquel, Thomas; Victoni, Tatiana; Fautrel, Alain; Robert, Sacha; Gleonnec, Florence; Guezingar, Marie; Couillin, Isabelle; Catros, Véronique; Boichot, Elisabeth; Lagente, Vincent

    2014-04-01

    Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury.

  16. Effect of adenosine and adenosine analogues on cyclic AMP accumulation in cultured mesangial cells and isolated glomeruli of the rat.

    PubMed Central

    Olivera, A.; Lopez-Novoa, J. M.

    1992-01-01

    1. Changes in intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were studied in rat isolated glomeruli and cultured glomerular mesangial cells exposed to adenosine and to the preferential A1 receptor agonist N6-R-1-methyl-2-phenylethyl adenosine (R-PIA), or the potent A2 adenosine receptor agonist 5-(N-ethylcarboxamide)adenosine (NECA). 2. Whereas NECA and adenosine triggered a dose-dependent increase in cyclic AMP values with EC50 values of approximately 10(-6) M and 3 x 10(-5) M respectively, R-PIA lowered cyclic AMP levels at concentrations of 10(-6) M or less and increased them at higher concentrations. 3. The time-course of the increase induced by 10(-6) M NECA was slower than that induced by 10(-4) M adenosine. Adenosine produced a maximal stimulation within the first minute, whereas the effect of NECA in both glomeruli and mesangial cells was noticeable only from the second minute of incubation. 4. The effects of the agonists R-PIA and NECA on the cyclic AMP system were blocked respectively by the A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthihe (DPCPX) at 10(-6) M and the A2 antagonist N-(2-dimethylaminoethyl)-N-methyl-4-(2, 3, 6, 7-tetrahydro-2,b-dioxo-1, 3-dipropyl-1H-purin-8-yl) benzene sulphonamide (PD115,199) at 10(-6) M. Theophylline, a known antagonist of adenosine receptors, inhibited the action of adenosine on cyclic AMP in mesangial cells. Dipyridamole, an inhibitor of the uptake of adenosine by the cells, enhanced the response to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1330173

  17. [The involvement of adenosine and adenosine deaminase in experimental myocardial infarct].

    PubMed

    Stratone, A; Busuioc, A; Roşca, V; Bazgan, L; Popa, M; Hăulică, I

    1989-01-01

    By the ligature of the left coronary artery in the rat anesthetized with nembutal (10 mg/100 i.p.) a significant increase of the 5'-nucleotidase activity (Wooton method) was noticed 10 minutes after the left ventricle infarction (from an average value of 1038.5 +/- 187 mU/g tissue to 1537 +/- 225 mU/g fresh tissue). The adenosine desaminase levels spectrophotometrically determined by Denstedt technique, do not appear significantly modified 10 or 30 minutes after the left ventricle infarction. The chromatographically determined adenosine levels, by HPLC technique, decrease from the average value of 11.63 +/- 1.4 micrograms/mg PT to 8.60 +/- 1.0 micrograms/mg PT 30 minutes after infarction. The observed changes are explained by the conditions of hypoxia in the infarcted ventricle which lead to the raise in adenosine levels by activating the 5'-nucleotidase and their depression by a very fast metabolism of the same substance.

  18. Adenosine and inflammation: what's new on the horizon?

    PubMed

    Antonioli, Luca; Csóka, Balázs; Fornai, Matteo; Colucci, Rocchina; Kókai, Endre; Blandizzi, Corrado; Haskó, György

    2014-08-01

    Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects. The scientific community is making intensive efforts to design novel adenosine receptor ligands endowed with greater selectivity or to develop innovative compounds acting as allosteric receptor modulators. In parallel, research is focusing on novel pharmacological entities (designated as adenosine-regulating agents) that can increase, in a site- and event-specific manner, adenosine concentrations at the inflammatory site, thereby minimizing the adverse systemic effects of adenosine.

  19. Rhodium Complex and Enzyme Couple Mediated Electrochemical Detection of Adenosine.

    PubMed

    Han, Dawoon; Kim, Hyeong-Mook; Chand, Rohit; Kim, Gyumin; Shin, Ik-Soo; Kim, Yong-Sang

    2015-10-01

    Adenosine is one of the nucleoside which plays an important role in signal transduction and neuromodulation. This work proposes a simple electrochemical assay, comprising two enzymes and rhodium complex based electron transfer mediator, for the detection of adenosine. Sequential reaction of adenosine deaminase and L-glutamic dehydrogenase and the supporting cycle between β-NADH and mediator enable quantitative analysis of adenosine. Role of electron transfer mediator is the conveyance of proton from electrode to β-NAD(+) for regeneration of β-NADH. The electrochemical characteristics of electron transfer mediator were also studied. Real-time adenosine detection was carried out using this multiple enzyme based chronoamperometric assay. The analysis results show a low limit of detection (140 μM) and good correspondence between current signal and the adenosine concentration (R (2) = 0.997).

  20. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors.

    PubMed

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32-35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR.

  1. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

    PubMed Central

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    2016-01-01

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

  2. Small molecule adenosine 5'-monophosphate activated protein kinase (AMPK) modulators and human diseases.

    PubMed

    Rana, Sandeep; Blowers, Elizabeth C; Natarajan, Amarnath

    2015-01-08

    Adenosine 5'-monophosphate activated protein kinase (AMPK) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy homeostasis. AMPK is a serine/threonine kinase that is activated by upstream kinases LKB1, CaMKKβ, and Tak1, among others. AMPK exists as αβγ trimeric complexes that are allosterically regulated by AMP, ADP, and ATP. Dysregulation of AMPK has been implicated in a number of metabolic diseases including type 2 diabetes mellitus and obesity. Recent studies have associated roles of AMPK with the development of cancer and neurological disorders, making it a potential therapeutic target to treat human diseases. This review focuses on the structure and function of AMPK, its role in human diseases, and its direct substrates and provides a brief synopsis of key AMPK modulators and their relevance in human diseases.

  3. Site of origin and mechanism of action of adenosine in the frog sympathetic ganglion

    SciTech Connect

    Bencherif, M.

    1987-01-01

    The contribution of pre and postsynaptic activation on the release of {sup 3}H-purines was studied in the isolated sympathetic paravertebral ganglion of the frog. Preganglionic stimulation induced an overall release of {sup 3}H-purines. This release is blocked by atropine and curare and can be induced by carbachol and antidromic stimulation. Analyses of the effluent by anion exchange chromatography and by HPLC showed that the non-nucleotide fractions constituted most of the counts released. Hence, nucleosides are the main products released by the ganglion and did not arise from hydrolysis of extracellular ATP. We studied the effect of synaptic activity on tritiated inositol release (IR). This release did not change during orthodromic stimulation. However, upon cessation of the stimulation, release increased rapidly and remained elevated for at least 45 minutes. This increase in IR was reduced by suffusion of the ganglia with either acetylcholine or adenosine.

  4. Non-infectious lung disease in patients with adenosine deaminase deficient severe combined immunodeficiency.

    PubMed

    Booth, C; Algar, V E; Xu-Bayford, J; Fairbanks, L; Owens, C; Gaspar, H B

    2012-06-01

    Adenosine deaminase deficiency is a disorder of purine metabolism manifesting severe combined immunodeficiency (ADA-SCID) and systemic abnormalities. Increased levels of the substrate deoxyadenosine triphosphate (dATP) lead to immunodeficiency and are associated in a murine model with pulmonary insufficiency. We compared a cohort of patients with ADA-SCID and X-linked SCID and found that despite similar radiological and respiratory findings, positive microbiology is significantly less frequent in ADA-SCID patients (p < 0.0005), suggesting a metabolic pathogenesis for the lung disease. Clinicians should be aware of this possibility and correct metabolic abnormalities either through enzyme replacement or haematopoietic stem cell transplant, in addition to treating infectious complications.

  5. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction.

    PubMed

    Giniatullin, A; Petrov, A; Giniatullin, R

    2015-01-29

    Adenosine 5'-triphosphate (ATP) is the main co-transmitter accompanying the release of acetylcholine from motor nerve terminals. Previously, we revealed the direct inhibitory action of extracellular ATP on transmitter release via redox-dependent mechanism. However, the receptor mechanism of ATP action and ATP-induced sources of reactive oxygen sources (ROS) remained not fully understood. In the current study, using microelectrode recordings of synaptic currents from the frog neuromuscular junction, we analyzed the receptor subtype involved in synaptic action of ATP, receptor coupling to NADPH oxidase and potential location of ATP receptors within the lipid rafts. Using subtype-specific antagonists, we found that the P2Y13 blocker 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde did not prevent the depressant action of ATP. In contrast, the P2Y12 antagonist 2-methylthioadenosine 5'-monophosphate abolished the inhibitory action of ATP, suggesting the key role of P2Y12 receptors in ATP action. As the action of ATP is redox-dependent, we also tested potential involvement of the NADPH oxidase, known as a common inducer of ROS. The depressant action of extracellular ATP was significantly reduced by diphenyleneiodonium chloride and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, two structurally different inhibitors of NADPH oxidase, indicating that this enzyme indeed mediates the action of ATP. Since the location and activity of various receptors are often associated with lipid rafts, we next tested whether ATP-driven inhibition depends on lipid rafts. We found that the disruption of lipid rafts with methyl-beta-cyclodextrin reduced and largely delayed the action of ATP. Taken together, these data revealed key steps in the purinergic control of synaptic transmission via P2Y12 receptors associated with lipid rafts, and identified NADPH oxidase as the main source of ATP-induced inhibitory ROS at the neuromuscular

  6. Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner.

    PubMed

    Cui, Mei; Ding, Hongyan; Chen, Fangzhe; Zhao, Yanxin; Yang, Qi; Dong, Qiang

    2016-01-01

    This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

  7. Turnover of adenosine in plasma of human and dog blood

    SciTech Connect

    Moeser, G.H.S.; Schrader, J.; Deussen, A.

    1989-04-01

    To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a ''stopping solution.'' Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added (/sup 3/H)adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not