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Sample records for enhancing extracellular atp

  1. Extracellular ATP

    PubMed Central

    Chivasa, Stephen; Tomé, Daniel FA; Murphy, Alex M; Hamilton, John M; Lindsey, Keith; Carr, John P

    2009-01-01

    Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to cxontrol gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.1 Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.2 A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death. PMID:20009563

  2. Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death. Results APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure. Conclusion We suggest that under pathological conditions

  3. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants1[OPEN

    PubMed Central

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-01-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg2+ as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. PMID:26224801

  4. Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor.

    PubMed

    Xu, Pengfei; Xu, Yongteng; Hu, Bin; Wang, Jue; Pan, Rui; Murugan, Madhuvika; Wu, Long-Jun; Tang, Yamei

    2015-11-01

    Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP-P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP-P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP-P2X7R in brain damage in RBI, suggesting that inhibition of ATP-P2X7R axis might be a potential strategy for the treatment of patients with RBI. PMID:26122280

  5. Extracellular ATP enhances radiation-induced brain injury through microglial activation and paracrine signaling via P2X7 receptor.

    PubMed

    Xu, Pengfei; Xu, Yongteng; Hu, Bin; Wang, Jue; Pan, Rui; Murugan, Madhuvika; Wu, Long-Jun; Tang, Yamei

    2015-11-01

    Activation of purinergic receptors by extracellular ATP (eATP) released from injured cells has been implicated in the pathogenesis of many neuronal disorders. The P2X7 receptor (P2X7R), an ion-selective purinergic receptor, is associated with microglial activation and paracrine signaling. However, whether ATP and P2X7R are involved in radiation-induced brain injury (RBI) remains to be determined. Here, we found that the eATP level was elevated in the cerebrospinal fluid (CSF) of RBI patients and was associated with the clinical severity of the disorder. In our experimental model, radiation treatment increased the level of eATP in the supernatant of primary cultures of neurons and glial cells and in the CSF of irradiated mice. In addition, ATP administration activated microglia, induced the release of the inflammatory mediators such as cyclooxygenase-2, tumor necrosis factor α and interleukin 6, and promoted neuronal apoptosis. Furthermore, blockade of ATP-P2X7R interaction using P2X7 antagonist Brilliant Blue G or P2X7 knockdown suppressed radiation-induced microglial activation and proliferation in the hippocampus, and restored the spatial memory of irradiated mice. Finally, we found that the PI3K/AKT and nuclear factor κB mediated pathways were downstream of ATP-P2X7R signaling in RBI. Taken together, our results unveiled the critical role of ATP-P2X7R in brain damage in RBI, suggesting that inhibition of ATP-P2X7R axis might be a potential strategy for the treatment of patients with RBI.

  6. Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

    PubMed Central

    Tarasiuk, A; Bar-Shimon, M; Gheber, L; Korngreen, A; Grossman, Y; Priel, Z

    1995-01-01

    Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP. Images FIGURE 6 PMID:7756536

  7. Intensification of ciliary motility by extracellular ATP.

    PubMed

    Ovadyahu, D; Eshel, D; Priel, Z

    1988-01-01

    Ciliary metachronism and motility were examined optically in tissue cultures from frog palate epithelium as a function of extracellular ATP concentration in the range of 10(-7)-10(-3) M. The main findings were: a) upon addition of ATP the metachronal wavelength increased by a factor of up to 2. b) the velocity of the metachronal wave increased by a factor of up to 5. c) the frequency of ciliary beating increased by a factor of up to 2-3, the increase being temperature insensitive in the range of 15 degrees C-25 degrees C. d) the area under the 1-second FFT spectrum decreased by a factor of up to 2.5. e) the energy of the metachronal wave is increased by a factor of up to 9.5. f) all the spectrum parameters are subject to influence by ATP, as also by ADP and AMP. However, there are pronounced differences in the various responses to them. Based on these findings, physical aspects of the rate increase of particle transport caused by addition of extracellular ATP are explained. A plausible overall chemical mechanism causing pronounced changes in ciliary motility is discussed.

  8. Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

    PubMed

    Weihs, Anna M; Fuchs, Christiane; Teuschl, Andreas H; Hartinger, Joachim; Slezak, Paul; Mittermayr, Rainer; Redl, Heinz; Junger, Wolfgang G; Sitte, Harald H; Rünzler, Dominik

    2014-09-26

    Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.

  9. Extracellular ATP protects endothelial cells against DNA damage.

    PubMed

    Aho, Joonas; Helenius, Mikko; Vattulainen-Collanus, Sanna; Alastalo, Tero-Pekka; Koskenvuo, Juha

    2016-09-01

    Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers γH2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.

  10. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

    PubMed Central

    Zhao, Ronglan; Liang, Dongchun; Sun, Deming

    2016-01-01

    Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases. PMID:27196432

  11. 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. PMID:8366007

  12. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport.

    PubMed

    Tang, Wenqiang; Brady, Shari R; Sun, Yu; Muday, Gloria K; Roux, Stanley J

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  13. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    NASA Technical Reports Server (NTRS)

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  14. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport.

    PubMed

    Tang, Wenqiang; Brady, Shari R; Sun, Yu; Muday, Gloria K; Roux, Stanley J

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed. PMID:12529523

  15. Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

    PubMed Central

    Sivaramakrishnan, Venketesh

    2015-01-01

    ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd3+ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd3+. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd3+, while NO donors rescued apyrase- and Gd3+-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd3+-sensitive receptor that is coupled with intracellular NO production. PMID:26048010

  16. Toll-Like Receptor-Triggered Calcium Mobilization Protects Mice against Bacterial Infection through Extracellular ATP Release

    PubMed Central

    Ren, Hua; Teng, Yunfei; Tan, Binghe; Zhang, Xiaoyu; Jiang, Wei; Liu, Mingyao; Jiang, Wenzheng

    2014-01-01

    Extracellular ATP (eATP), released as a “danger signal” by injured or stressed cells, plays an important role in the regulation of immune responses, but the relationship between ATP release and innate immune responses is still uncertain. In this study, we demonstrated that ATP was released through Toll-like receptor (TLR)-associated signaling in both Escherichia coli-infected mice and lipopolysaccharide (LPS)- or Pam3CSK4-treated macrophages. This ATP release could be blocked completely only by N-ethylmaleimide (NEM), not by carbenoxolone (CBX), flufenamic acid (FFA), or probenecid, suggesting the key role of exocytosis in this process. Furthermore, LPS-induced ATP release could also be reduced dramatically through suppressing calcium mobilization by use of U73122, caffeine, and thapsigargin (TG). In addition, the secretion of interleukin-1β (IL-1β) and CCL-2 was enhanced significantly by ATP, in a time- and dose-dependent manner. Meanwhile, macrophage-mediated phagocytosis of bacteria was also promoted significantly by ATP stimulation. Furthermore, extracellular ATP reduced the number of invading bacteria and protected mice from peritonitis by activating purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was overtly increased by ATP in antibacterial immune responses. Accordingly, if we blocked the P2X- and P2Y-associated signaling pathway by using suramin and pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid), tetrasodium salt (PPADS), the ATP-enhanced immune response was restrained significantly. Taken together, our findings reveal an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of calcium mobilization-mediated ATP release in infectious diseases. PMID:25245808

  17. Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

    PubMed

    Chara, Osvaldo; Pafundo, Diego E; Schwarzbaum, Pablo J

    2010-06-21

    A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

  18. Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells

    PubMed Central

    Korngreen, Alon; Ma, Weiyuan; Priel, Zvi; Silberberg, Shai D

    1998-01-01

    A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole-cell and outside-out patch configurations of the patch-clamp technique. In solutions designed to maximize currents through voltage-gated calcium channels, there were no indications of voltage-gated Ba2+ currents. Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible. Including GDP-β-S in the intracellular solution did not inhibit the effects of ATP, nor did GTP-γ-S irreversibly activate the conductance. In outside-out membrane patches, with GDP-β-S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at −120 mV. Suramin (100 μM) inhibited the whole-cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP-activated channels in outside-out membrane patches. Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At −40 mV, the time to half-maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP-β-S in the intracellular solution. The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4-fold longer than t½ at −40 mV. Half-maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP

  19. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain.

    PubMed

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  20. Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain

    PubMed Central

    Masuda, Takahiro; Ozono, Yui; Mikuriya, Satsuki; Kohro, Yuta; Tozaki-Saitoh, Hidetoshi; Iwatsuki, Ken; Uneyama, Hisayuki; Ichikawa, Reiko; Salter, Michael W.; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Activation of purinergic receptors in the spinal cord by extracellular ATP is essential for neuropathic hypersensitivity after peripheral nerve injury (PNI). However, the cell type responsible for releasing ATP within the spinal cord after PNI is unknown. Here we show that PNI increases expression of vesicular nucleotide transporter (VNUT) in the spinal cord. Extracellular ATP content ([ATP]e) within the spinal cord was increased after PNI, and this increase was suppressed by exocytotic inhibitors. Mice lacking VNUT did not show PNI-induced increase in [ATP]e and had attenuated hypersensitivity. These phenotypes were recapitulated in mice with specific deletion of VNUT in spinal dorsal horn (SDH) neurons, but not in mice lacking VNUT in primary sensory neurons, microglia or astrocytes. Conversely, ectopic VNUT expression in SDH neurons of VNUT-deficient mice restored PNI-induced increase in [ATP]e and pain. Thus, VNUT is necessary for exocytotic ATP release from SDH neurons which contributes to neuropathic pain. PMID:27515581

  1. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

    SciTech Connect

    Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  2. Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction

    PubMed Central

    Giniatullin, AR; Grishin, SN; Sharifullina, ER; Petrov, AM; Zefirov, AL; Giniatullin, RA

    2005-01-01

    During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2′,7′-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings. PMID:15774519

  3. The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation.

    PubMed

    Wakizoe, Erika; Ashizawa, Koji; Sakamoto, Shinsuke H; Hemmi, Koichiro; Kobayashi, Ikuo; Tsuzuki, Yasuhiro

    2015-12-01

    We measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: 6 mm in diameter). Then, the effects of pre-treatment (100 μM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (-) PPADS and in the presence (+) or absence (-) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(-) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(-) RB2(-) and ATP(+)RB2(-) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(-) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(-) RB2(-) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

  4. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

    PubMed Central

    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  5. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

    PubMed Central

    Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  6. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  7. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    PubMed

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

  8. Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism.

    PubMed

    Kimball, B C; Yule, D I; Mulholland, M W

    1996-04-01

    In the myenteric plexus, ATP is released as a neurotransmitter by "purinergic" nerves, relaxing visceral smooth muscle. We report a signal transduction mechanism for ATP in cultured myenteric neurons involving receptor-mediated release of intracellular Ca2+ stores. Primary cultures of myenteric neurons from guinea pigs taenia coli were loaded with the Ca2+ indicator fura 2-acetoxymethyl ester (AM) and examined using digital imaging microscopy. Superfusion of single neurons with ATP (0.01-1,000 microM) resulted in concentration-dependent increases in intracellular Ca2+ concentration ([Ca2+]i) that were independent of extracellular Ca2+. Decrements in peak [Ca2+]i were seen with repetitive ATP exposure. Responsiveness of myenteric neurons to purinergic agonists (100 microM) was consistent with action at a neuronal P 2y purinoceptor: 2-chloro-ATP = ATP = 2-methyl-thio-ATP (MeSATP) > ADP > alpha, beta-MeATP = beta,gamma-MeATP > AMP > adenosine. ATP-evoked Ca2+ transients were inhibited dose dependently by suramin, a nonspecific P2 antagonist, and reactive blue 2, a specific P 2y antagonist. ATP and cyclopiazonic acid (30 microM) appear to release an identical intracellular Ca2+ store. Preincubation with the aminosteroid U-73122 (10 microM) inhibited ATP-evoked Ca2+ transients by 71 +/- 7%, whereas phorbol ester pretreatment (phorbol 12-myristate 13-acetate, 100 nM, 5 min) caused a 76 +/- 4% inhibition. Peak [Ca2+]i evoked by ATP was not affected by preincubation with pertussis toxin (100 ng/ml, 24 h) or nifedipine (10 microM). These data suggest a signal transduction mechanism for ATP in cultured myenteric neurons involving purinoceptor-mediated activation of phospholipase C (PLC), with release of D-myo-inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores.

  9. Kinetics of extracellular ATP from goldfish hepatocytes: a lesson from mathematical modeling.

    PubMed

    Chara, Osvaldo; Pafundo, Diego E; Schwarzbaum, Pablo J

    2009-07-01

    In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220-R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J(L)) and nonlytic mechanisms (J(NL)), ATPe diffusion (J(D)), and ATPe consumption by ectonucleotidases (J(V)). Particular focus was given to J(NL), defined as the product of a time function (J(R)) and a positive feedback mechanism whereby ATPe amplifies J(NL). Several J (R) functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that: - use of J(D) does not lead to improvements of the models. - Constant and Step time functions are only applicable when J(R)=0 (and thus, J(NL)=0), so that the only source of ATPe would be J(L), a result incompatible with experimental data. - use of impulse, Gaussian, and lognormal J(R)s in the models led to reasonable good fits to experimental data, with the lognormal function in model 1 providing the best option. Finally, the predictive nature of model 1 loaded with a lognormal J(R) was tested by simulating different putative in vivo scenarios where J(V) and J(NL) were varied over ample ranges.

  10. Extracellular ATP signaling via P2X(4) receptor and cAMP/PKA signaling mediate ATP oscillations essential for prechondrogenic condensation.

    PubMed

    Kwon, Hyuck Joon

    2012-09-01

    Prechondrogenic condensation is the most critical process in skeletal patterning. A previous study demonstrated that ATP oscillations driven by Ca(2+) oscillations play a critical role in prechondrogenic condensation by inducing oscillatory secretion. However, it remains unknown what mechanisms initiate the Ca(2+)-driven ATP oscillations, mediate the link between Ca(2+) and ATP oscillations, and then result in oscillatory secretion in chondrogenesis. This study has shown that extracellular ATP signaling was required for both ATP oscillations and prechondrogenic condensation. Among P2 receptors, the P2X(4) receptor revealed the strongest expression level and mediated ATP oscillations in chondrogenesis. Moreover, blockage of P2X(4) activity abrogated not only chondrogenic differentiation but also prechondrogenic condensation. In addition, both ATP oscillations and secretion activity depended on cAMP/PKA signaling but not on K(ATP) channel activity and PKC or PKG signaling. This study proposes that Ca(2+)-driven ATP oscillations essential for prechondrogenic condensation is initiated by extracellular ATP signaling via P2X(4) receptor and is mediated by cAMP/PKA signaling and that cAMP/PKA signaling induces oscillatory secretion to underlie prechondrogenic condensation, in cooperation with Ca(2+) and ATP oscillations.

  11. Modulation of Root Skewing in Arabidopsis by Apyrases and Extracellular ATP.

    PubMed

    Yang, Xingyan; Wang, Bochu; Farris, Ben; Clark, Greg; Roux, Stanley J

    2015-11-01

    When plant primary roots grow along a tilted surface that is impenetrable, they can undergo a slanted deviation from the direction of gravity called skewing. Skewing is induced by touch stimuli which the roots experience as they grow along the surface. Touch stimuli also induce the release of extracellular ATP (eATP) into the plant's extracellular matrix, and two apyrases (NTPDases) in Arabidopsis, APY1 and APY2, can help regulate the concentration of eATP. The primary roots of seedlings overexpressing APY1 show less skewing than wild-type plants. Plants suppressed in their expression of APY1 show more skewing than wild-type plants. Correspondingly, chemical inhibition of apyrase activity increased skewing in mutants and wild-type roots. Exogenous application of ATP or ATPγS also increased skewing in wild-type roots, which could be blocked by co-incubation with a purinergic receptor antagonist. These results suggest a model in which gradients of eATP set up by differential touch stimuli along roots help direct skewing in roots growing along an impenetrable surface.

  12. Neuronal NTPDase3 Mediates Extracellular ATP Degradation in Trigeminal Nociceptive Pathway

    PubMed Central

    Ma, Lihua; Trinh, Thu; Ren, Yanfang; Dirksen, Robert T.; Liu, Xiuxin

    2016-01-01

    ATP induces pain via activation of purinergic receptors in nociceptive sensory nerves. ATP signaling is terminated by ATP hydrolysis mediated by cell surface-localized ecto-nucleotidases. Using enzymatic histochemical staining, we show that ecto-ATPase activity is present in mouse trigeminal nerves. Using immunofluorescence staining, we found that ecto-NTPDase3 is expressed in trigeminal nociceptive neurons and their projections to the brainstem. In addition, ecto-ATPase activity and ecto-NTPDase3 are also detected in the nociceptive outermost layer of the trigeminal subnucleus caudalis. Furthermore, we demonstrate that incubation with anti-NTPDase3 serum reduces extracellular ATP degradation in the nociceptive lamina of both the trigeminal subnucleus caudalis and the spinal cord dorsal horn. These results are consistent with neuronal NTPDase3 activity modulating pain signal transduction and transmission by affecting extracellular ATP hydrolysis within the trigeminal nociceptive pathway. Thus, disruption of trigeminal neuronal NTPDase3 expression and localization to presynaptic terminals during chronic inflammation, local constriction and injury may contribute to the pathogenesis of orofacial neuropathic pain. PMID:27706204

  13. Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

    PubMed Central

    Medina-Castellanos, Elizabeth; Esquivel-Naranjo, Edgardo U.; Heil, Martin; Herrera-Estrella, Alfredo

    2014-01-01

    The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals. PMID:25484887

  14. LMO4 mRNA stability is regulated by extracellular ATP in F11 cells

    SciTech Connect

    Chen, Hsiao-Huei . E-mail: hchen@uottawa.ca; Xu, Jin; Safarpour, Farzaneh; Stewart, Alexandre F.R.

    2007-05-25

    LIM only domain protein 4 (LMO4) interacts with many signaling and transcription factors to regulate cellular proliferation, differentiation and plasticity. In Drosophila, mutations in the 3' untranslated region (UTR) of the homologue dLMO cause a gain of function by increasing mRNA stability. LMO4 3'UTR contains several AU-rich elements (ARE) and is highly conserved among vertebrates, suggesting that RNA destabilizing mechanisms are evolutionarily conserved. Here, we found that extracellular ATP stabilized LMO4 mRNA in F11 cells. The LMO4 3'UTR added to a luciferase reporter markedly reduced reporter activity under basal conditions, but increased activity with ATP treatment. Two ARE motifs were characterized in the LMO4 3'UTR. ATP increased binding of HuD protein to ARE1. ARE1 conferred ATP and HuD-dependent mRNA stabilization. In contrast, sequences flanking ARE2 bound CUGBP1 and ATP destabilized this complex. Thus, our results suggest that ATP modulates recruitment of RNA-binding proteins to the 3'UTR to stabilize LMO4 mRNA.

  15. Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation.

    PubMed

    Orriss, Isabel R; Key, Michelle L; Hajjawi, Mark O R; Arnett, Timothy R

    2013-01-01

    Previous studies have shown that exogenous ATP (>1 µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP(i)). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5 U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2 P(i)). Addition of 0.5 U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5 U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP(i)-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.

  16. Transcriptional regulation of IL-6 in bile duct epithelia by extracellular ATP

    PubMed Central

    Yu, Jin; Sheung, Nina; Soliman, Elwy M.; Spirli, Carlo; Dranoff, Jonathan A.

    2009-01-01

    The inflammatory cytokine IL-6 is essential for cell survival after liver injury. Bile duct epithelia (BDE) markedly upregulate IL-6 release after liver injury, but the mechanisms regulating this have not been defined. Purinergic signals induce multiple potent downstream effects in BDE, so the goals of this study were to determine whether extracellular ATP regulates BDE IL-6 transcription and to identify the molecular mechanisms regulating this process. Effects of extracellular nucleotides on IL-6 transcription in primary rat bile duct epithelia were assessed. The relative effects of cAMP and cytosolic calcium were determined by use of agonists and antagonists. The role of the cAMP response element (CRE) was determined by site-directed mutagenesis. We found that ATP potently upregulated IL-6 mRNA, and that the pharmacological profile for IL-6 upregulation was most consistent with the newly identified P2Y11 receptor. This occurred in a cAMP-dependent and calcium-dependent fashion. The effect of cAMP and calcium agonists on IL-6 promoter activity was synergistic, and mutation of the IL-6 CRE blocked upregulation by ATP. Taken together, these data show that extracellular ATP acts through a mechanism involving a rat P2Y receptor functionally related to the P2Y11 receptor, cAMP, and calcium signals and that the IL-6 promoter CRE to upregulate transcription of IL-6 in BDE. Since IL-6 has such critical importance in the liver, it is likely that this pathway is of great relevance to the understanding of hepatic response to injury. PMID:19136380

  17. ATP is released from autophagic vesicles to the extracellular space in a VAMP7-dependent manner.

    PubMed

    Fader, Claudio Marcelo; Aguilera, Milton Osmar; Colombo, María Isabel

    2012-12-01

    Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome. SNAREs proteins are key molecules of the vesicle fusion machinery. Our results indicate that in a mammalian tumor cell line a subset of VAMP7 (V-SNARE)-positive vacuoles colocalize with LC3 at the cell periphery (focal adhesions) upon starvation. The re-distribution of VAMP7 positive structures is a microtubule-dependent event, with the participation of the motor protein KIF5 and the RAB7 effector RILP. Interestingly, most of the VAMP7-labeled vesicles were loaded with ATP. Moreover, in cells subjected to starvation, these structures fuse with the plasma membrane to release the nucleotide to the extracellular medium. Summarizing, our results show the molecular components involved in the release of ATP to extracellular space, which is recognized as an important autocrine/paracrine signal molecule that participates in the regulation of several cellular functions such as immunogenicity of cancer cell death or inflammation.

  18. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  19. Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

    PubMed

    Li, Shuo; Peng, Weijiao; Chen, Xiaoli; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-08-01

    Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP

  20. Extracellular ATP stimulates exocytosis via localized Ca(2+) release from acidic stores in rat pancreatic beta cells.

    PubMed

    Xie, Li; Zhang, Ming; Zhou, Wei; Wu, Zhengxing; Ding, Jiuping; Chen, Liangyi; Xu, Tao

    2006-04-01

    Three different methods, membrane capacitance (C(m)) measurement, amperometry and FM dye labeling were used to investigate the role of extracellular ATP in insulin secretion from rat pancreatic beta cells. We found that extracellular application of ATP mobilized intracellular Ca(2+) stores and synchronously triggered vigorous exocytosis. No influence of ATP on the readily releasable pool of vesicles was observed, which argues against a direct modulation of the secretory machinery at a level downstream of Ca(2+) elevation. The stimulatory effects of ATP were greatly reduced by intracellular perfusion of BAPTA but not EGTA, suggesting a close spatial association of fusion sites with intracellular Ca(2+) releasing sites. ATP-induced Ca(2+) transients and exocytosis were not blocked by thapsigargin (TG), by a ryanodine receptor antagonist or by dissipation of pH in acidic stores by monensin alone, but they were greatly attenuated by IP(3) receptor inhibition as well as ionomycin plus monensin, suggesting involvement of IP(3)-sensitive acidic Ca(2+) stores. Taken together, our data suggest that extracellular ATP triggers exocytosis by mobilizing spatially limited acidic Ca(2+) stores through IP(3) receptors. This mechanism may explain how insulin secretion from the pancreas is coordinated through diffusible ATP that is co-released with insulin. PMID:16536741

  1. Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 CellsV⃞

    PubMed Central

    Morelli, Anna; Chiozzi, Paola; Chiesa, Anna; Ferrari, Davide; Sanz, Juana M.; Falzoni, Simonetta; Pinton, Paolo; Rizzuto, Rosario; Olson, Michael F.; Di Virgilio, Francesco

    2003-01-01

    The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I. PMID:12857854

  2. Vaccination with Antigen Combined with αβ-ATP as a Vaccine Adjuvant Enhances Antigen-Specific Antibody Production via Dendritic Cell Activation.

    PubMed

    Matsuo, Kazuhiko; Nishiuma, Satoshi; Hasegawa, Yuta; Kawabata, Fumika; Kitahata, Kosuke; Nakayama, Takashi

    2016-01-01

    Adjuvants are required to enhance antigen-specific immune responses by vaccines. Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X and P2Y receptors and triggers the activation of dendritic cells (DCs). Here we investigated the in vivo adjuvant efficacy of α,β-methylene-ATP (αβ-ATP), a non-hydrolysable form of ATP. We found that intradermal injection of ovalbumin (OVA), as a model antigen, combined with αβ-ATP, as the adjuvant, enhanced OVA-specific immune responses more than OVA alone. Additionally, DCs in the skin of mice injected with OVA and αβ-ATP had increased expression of major histocompatibility complex class II and co-stimulator molecules, CD40, CD80, and CD86, suggesting that αβ-ATP activated DC. These findings indicate that αβ-ATP functions as a potent vaccine adjuvant. PMID:27251512

  3. Extracellular ATP is cytotoxic to mononuclear phagocytes but does not induce killing of intracellular Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Woo, Seng-Ryong; Barletta, Raúl G; Czuprynski, Charles J

    2007-09-01

    Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing of Mycobacterium species in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability of M. avium subsp. paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP to M. avium subsp. paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival of M. avium subsp. paratuberculosis in bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival of M. avium subsp. paratuberculosis, nor were the numbers of viable Mycobacterium avium subsp. avium or Mycobacterium bovis BCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellular M. avium subsp. paratuberculosis in bovine mononuclear phagocytes.

  4. The modulation of haemolymph arginine kinase on the extracellular ATP induced bactericidal immune responses in the Pacific oyster Crassostrea gigas.

    PubMed

    Jiang, Shuai; Jia, Zhihao; Chen, Hao; Wang, Lingling; Song, Linsheng

    2016-07-01

    Arginine kinase is an important phosphagen kinase (PK) which plays an essential role in ATP buffering systems in invertebrates. In the present study, an arginine kinase (designated CgAK) was isolated by the lipopolysaccharide (LPS) affinity chromatography from the haemolymph of Crassostrea gigas. CgAK could directly bind to LPS in a concentration-dependent manner with the dissociation constant (Kd) of 2.46 × 10(-6) M. The interaction with LPS significantly decreased the ATP hydrolytic activity of CgAK, which in turn lead to the accumulation of ATP in vitro. The extracellular ATP stimulation could induce Ca(2+) influx, reactive oxygen species (ROS) production, and the release of lysosomal enzyme in the cellular immune response. In addition, ATP stimulation provoked the bactericidal activity towards Escherichia coli, and the scavenging ROS with N-acetyl-l-cysteine (NAC) abrogated the bactericidal activity, indicating that ATP stimulation could induce ROS-dependent antimicrobial activity in haemocytes. Collectively, the results demonstrated that the haemolymph CgAK could serve as an important purinergic regulator to modulate extracellular ATP, which might further have an important effect on the purinergic signaling-activated innate immune response of oyster.

  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. Characterization of the cytotoxic effect of extracellular ATP in J774 mouse macrophages.

    PubMed Central

    Murgia, M; Pizzo, P; Steinberg, T H; Di Virgilio, F

    1992-01-01

    Extracellular ATP (ATPo) is known to be cytotoxic to many cell types through a mechanism which is largely unknown. Very recently this nucleotide has been shown to cause cell death by apoptosis, probably by interacting with specific cell-surface receptors. In the present study we have investigated the mechanism of ATPo-dependent cytotoxicity in the macrophage-like mouse cell line J774. It has been previously reported that in this cell type ATPo activates trans-membrane Ca2+ and Na+ fluxes and a drastic increase in the plasma-membrane permeability to hydrophilic solutes smaller than 900 Da. These changes are followed by cell swelling and lysis. We show in the present study that, although this nucleotide triggers a rise in the cytoplasmic Ca2+ concentration, neither cell swelling nor lysis is Ca(2+)-dependent. Furthermore, cell lysis is not dependent on Na+ influx, as it is not prevented by iso-osmotic replacement of extracellular Na+ with choline or N-methylglucamine. On the contrary, ATPo-dependent cytotoxicity, but not the ATPo-dependent increase in plasma-membrane permeability, is completely abrogated in sucrose medium. Under our experimental conditions ATPo does not cause DNA fragmentation in J774 cells. We conclude from these findings that ATPo does not cause apoptosis of J774 macrophages and promotes a Ca(2+)- and Na(+)-independent colloido-osmotic lysis. Images Fig. 4. Fig. 7. PMID:1472003

  7. Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

    PubMed

    Brisevac, Dusica; Adzic, Marija; Laketa, Danijela; Parabucki, Ana; Milosevic, Milena; Lavrnja, Irena; Bjelobaba, Ivana; Sévigny, Jean; Kipp, Markus; Nedeljkovic, Nadezda

    2015-11-01

    Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis. PMID:26080748

  8. Inosine preserves ATP during ischemia and enhances recovery during reperfusion.

    PubMed

    Devous, M D; Lewandowski, E D

    1987-11-01

    The effects of exogenous inosine (IN) on high-energy phosphate metabolism and function in isolated, working rabbit hearts were monitored with 31P-nuclear magnetic resonance spectroscopy. Dynamic measurements of ATP and phosphocreatine (PCr) were made along with concomitant functional recordings during normal perfusion, global ischemia (IS), and reperfusion (RE). We found that 0.1 mM IN enhanced the rate of pressure development (dP/dt) within the left ventricle by 10 +/- 5% (n = 7). Although IN levels in treated hearts were elevated during normal perfusion, no effect was observed on ATP or PCr levels. However during IS, pretreatment with IN minimized ATP loss for the first 20 min relative to untreated controls (UNT, P less than 0.05). Both IN and UNT hearts that were ischemic for only 13.5 min regained function during a 60-min RE period. However, at the end of IS, IN hearts (n = 8) displayed 88 +/- 10% of the pre-IS ATP levels, whereas UNT hearts (n = 7) retained only 60 +/- 10%. With RE, ATP in IN hearts remained elevated over that of UNT hearts for the entire 60 min. IN treatment also increased the rate of recovery of dP/dt and maintained improved function over 60 min of RE. No correlation was found between post-IS ATP levels and dP/dt values during RE in either IN or UNT hearts. These data indicate that IN was protective against ATP loss during IS and improved functional recovery on RE.

  9. ATP-enhanced peroxidase-like activity of gold nanoparticles.

    PubMed

    Shah, Juhi; Purohit, Rahul; Singh, Ragini; Karakoti, Ajay Singh; Singh, Sanjay

    2015-10-15

    Gold nanoparticles (AuNPs) are known to possess intrinsic biological peroxidase-like activity that has applications in development of numerous biosensors. The reactivity of the Au atoms at the surface of AuNPs is critical to the performance of such biosensors, yet little is known about the effect of biomolecules and ions on the peroxidase-like activity. In this work, the effect of ATP and other biologically relevant molecules and ions over peroxidase-like activity of AuNPs are described. Contrary to the expectation that nanoparticles exposed to biomolecules may lose the catalytic property, ATP and ADP addition enhanced the peroxidase-like activity of AuNPs. The catalytic activity was unaltered by the addition of free phosphate, sulphate and carbonate anions however, addition of ascorbic acid to the reaction mixture diminished the intrinsic peroxidase-like activity of AuNPs, even in the presence of ATP and ADP. In contrast to AuNPs, ATP did not synergize and improve the peroxidase activity of the natural peroxidase enzyme, horseradish peroxidase.

  10. 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

  11. Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.

    PubMed

    Pant, H C; Terakawa, S; Yoshioka, T; Tasaki, I; Gainer, H

    1979-01-01

    Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.

  12. Exercise sensitizes skeletal muscle to extracellular ATP for IL-6 expression in mice.

    PubMed

    Fernández-Verdejo, R; Casas, M; Galgani, J E; Jaimovich, E; Buvinic, S

    2014-04-01

    Active skeletal muscle synthesizes and releases interleukin-6 (IL-6), which plays important roles in the organism's adaptation to exercise. Autocrine/paracrine ATP signaling has been shown to modulate IL-6 expression. The aim of this study was to determine whether a period of physical activity modifies the ATP-induced IL-6 expression. BalbC mice were either subject to 5 weeks voluntary wheel running (VA) or kept sedentary (SED). Flexor digitorum brevis muscles were dissected, stimulated with different ATP concentrations (0-100 μM) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10 μM ATP. In addition, there were lower P2Y2- and higher P2Y14-receptor mRNA levels in skeletal muscles of VA compared to SED mice, showing plasticity of nucleotide receptors with exercise. These results suggest that exercise increases skeletal muscle ATP sensitivity, a response dependent on the level of physical activity performed. This could have an important role in the mechanisms controlling skeletal muscle adaptation to exercise and training.

  13. Exercise sensitizes skeletal muscle to extracellular ATP for IL-6 expression in mice.

    PubMed

    Fernández-Verdejo, R; Casas, M; Galgani, J E; Jaimovich, E; Buvinic, S

    2014-04-01

    Active skeletal muscle synthesizes and releases interleukin-6 (IL-6), which plays important roles in the organism's adaptation to exercise. Autocrine/paracrine ATP signaling has been shown to modulate IL-6 expression. The aim of this study was to determine whether a period of physical activity modifies the ATP-induced IL-6 expression. BalbC mice were either subject to 5 weeks voluntary wheel running (VA) or kept sedentary (SED). Flexor digitorum brevis muscles were dissected, stimulated with different ATP concentrations (0-100 μM) and IL-6 mRNA levels were measured using qPCR. ATP evoked a concentration-dependent rise in IL-6 mRNA in both SED and VA mice. VA mice however, had significantly higher ATP sensitivity (pD2 pharmacological values: VA=5.58±0.02 vs. SED=4.95±0.04, p<0.05). Interestingly, in VA mice we observed a positive correlation between the level of physical activity and the IL-6 mRNA increase following fiber stimulation with 10 μM ATP. In addition, there were lower P2Y2- and higher P2Y14-receptor mRNA levels in skeletal muscles of VA compared to SED mice, showing plasticity of nucleotide receptors with exercise. These results suggest that exercise increases skeletal muscle ATP sensitivity, a response dependent on the level of physical activity performed. This could have an important role in the mechanisms controlling skeletal muscle adaptation to exercise and training. PMID:24022572

  14. Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca(2+) signals and an IL-6 autocrine loop.

    PubMed

    Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique; Buvinic, Sonja

    2014-04-15

    Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

  15. Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca2+ signals and an IL-6 autocrine loop

    PubMed Central

    Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique

    2014-01-01

    Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop. PMID:24518675

  16. Electrical stimulation induces IL-6 in skeletal muscle through extracellular ATP by activating Ca(2+) signals and an IL-6 autocrine loop.

    PubMed

    Bustamante, Mario; Fernández-Verdejo, Rodrigo; Jaimovich, Enrique; Buvinic, Sonja

    2014-04-15

    Interleukin-6 (IL-6) is an important myokine that is highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation, concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and p-STAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10-fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody or preincubation with the STAT3 inhibitor VIII reduced STAT3 activation evoked by extracellular ATP by 70%. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of the IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop. PMID:24518675

  17. Extracellular ATP and nitric oxide signaling pathways regulate redox-dependent responses associated to root hair growth in etiolated Arabidopsis seedlings

    PubMed Central

    Terrile, María Cecilia; Tonón, Claudia Virginia; Iglesias, María José; Lamattina, Lorenzo

    2010-01-01

    Extracellular ATP (eATP) and nitric oxide (NO) have emerged as crucial players in plant development, stress responses and cell viability. Glutathione (GSH) is an abundant reducing agent with proposed roles in plant growth, development and stress physiology. In a recent publication, we demonstrated that eATP and NO restore hypocotyl elongation of etiolated Arabidopsis seedlings treated with GSH. Here it is reported that exogenous ATP also restores root hair growth suggesting a role for ATP and NO in the regulation of redox balance associated to specific processes of plant morphogenesis. A tentative model integrating redox-, eATP- and NO-signaling pathways during root hair growth in Arabidopsis seedlings is presented. PMID:20404565

  18. Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

    PubMed Central

    2012-01-01

    Background Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11). Conclusions The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway. PMID:22420332

  19. 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

  20. Apyrase Suppression Raises Extracellular ATP Levels and Induces Gene Expression and Cell Wall Changes Characteristic of Stress Responses1[C][W][OPEN

    PubMed Central

    Lim, Min Hui; Wu, Jian; Yao, Jianchao; Gallardo, Ignacio F.; Dugger, Jason W.; Webb, Lauren J.; Huang, James; Salmi, Mari L.; Song, Jawon; Clark, Greg; Roux, Stanley J.

    2014-01-01

    Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes. PMID:24550243

  1. Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria.

    PubMed

    Albert, Lynal S; Brown, Derick G

    2015-08-01

    In this study we investigated the relationship between a rapid change in extracellular pH and the alteration of bacterial ATP concentration. This relationship is a key component of a hypothesis indicating that bacterial bioenergetics - the creation of ATP from ADP via a proton gradient across the cytoplasmic membrane - can be altered by the physiochemical charge-regulation effect, which results in a pH shift at the bacteria's surface upon adhesion to another surface. The bacterial ATP concentration was measured during a rapid change in extracellular pH from a baseline pH of 7.2 to pH values between 3.5 and 10.5. Experiments were conducted with four neutrophilic bacterial strains, including the Gram-negative Escherichia coli and Pseudomonas putida and the Gram-positive Bacillus subtilis and Staphylococcus epidermidis. A change in bulk pH produced an immediate response in bacterial ATP, demonstrating a direct link between changes in extracellular pH and cellular bioenergetics. In general, the shifts in ATP were similar across the four bacterial strains, with results following an exponential relationship between the extracellular pH and cellular ATP concentration. One exception occurred with S. epidermidis, where there was no variation in cellular ATP at acidic pH values, and this finding is consistent with this species' ability to thrive under acidic conditions. These results provide insight into obtaining a desired bioenergetic response in bacteria through (i) the application of chemical treatments to vary the local pH and (ii) the selection and design of surfaces resulting in local pH modification of attached bacteria via the charge-regulation effect.

  2. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

    PubMed

    Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

  3. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

    PubMed Central

    Díaz-Vegas, Alexis; Campos, Cristian A.; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

  4. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

    PubMed

    Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

  5. Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

    PubMed

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies.

  6. Electrical Stimuli Are Anti-Apoptotic in Skeletal Muscle via Extracellular ATP. Alteration of This Signal in Mdx Mice Is a Likely Cause of Dystrophy

    PubMed Central

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

  7. Electrical stimuli are anti-apoptotic in skeletal muscle via extracellular ATP. Alteration of this signal in Mdx mice is a likely cause of dystrophy.

    PubMed

    Valladares, Denisse; Almarza, Gonzalo; Contreras, Ariel; Pavez, Mario; Buvinic, Sonja; Jaimovich, Enrique; Casas, Mariana

    2013-01-01

    ATP signaling has been shown to regulate gene expression in skeletal muscle and to be altered in models of muscular dystrophy. We have previously shown that in normal muscle fibers, ATP released through Pannexin1 (Panx1) channels after electrical stimulation plays a role in activating some signaling pathways related to gene expression. We searched for a possible role of ATP signaling in the dystrophy phenotype. We used muscle fibers from flexor digitorum brevis isolated from normal and mdx mice. We demonstrated that low frequency electrical stimulation has an anti-apoptotic effect in normal muscle fibers repressing the expression of Bax, Bim and PUMA. Addition of exogenous ATP to the medium has a similar effect. In dystrophic fibers, the basal levels of extracellular ATP were higher compared to normal fibers, but unlike control fibers, they do not present any ATP release after low frequency electrical stimulation, suggesting an uncoupling between electrical stimulation and ATP release in this condition. Elevated levels of Panx1 and decreased levels of Cav1.1 (dihydropyridine receptors) were found in triads fractions prepared from mdx muscles. Moreover, decreased immunoprecipitation of Cav1.1 and Panx1, suggest uncoupling of the signaling machinery. Importantly, in dystrophic fibers, exogenous ATP was pro-apoptotic, inducing the transcription of Bax, Bim and PUMA and increasing the levels of activated Bax and cytosolic cytochrome c. These evidence points to an involvement of the ATP pathway in the activation of mechanisms related with cell death in muscular dystrophy, opening new perspectives towards possible targets for pharmacological therapies. PMID:24282497

  8. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    PubMed Central

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

    2016-01-01

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses in Arabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein, Camelina sativa lectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space group C222 or C2221, with unit-cell parameters a = 94.7, b = 191.5, c = 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants. PMID:27710944

  9. CD73 and AMPD3 deficiency enhance metabolic performance via erythrocyte ATP that decreases hemoglobin oxygen affinity

    PubMed Central

    O’Brien III, William G.; Berka, Vladimir; Tsai, Ah-Lim; Zhao, Zhaoyang; Lee, Cheng Chi

    2015-01-01

    Erythrocytes are the key target in 5′-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3−/− mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism. PMID:26249166

  10. Increased levels of extracellular ATP in glaucomatous retinas: Possible role of the vesicular nucleotide transporter during the development of the pathology

    PubMed Central

    Pérez de Lara, María J.; Guzmán-Aránguez, Ana; de la Villa, Pedro; Díaz-Hernández, Juan Ignacio; Miras-Portugal, María Teresa

    2015-01-01

    Purpose To study retinal extracellular ATP levels and to assess the changes in the vesicular nucleotide transporter (VNUT) expression in a murine model of glaucoma during the development of the disease. Methods Retinas were obtained from glaucomatous DBA/2J mice at 3, 9, 15, and 22 months together with C57BL/6J mice used as age-matched controls. To study retinal nucleotide release, the retinas were dissected and prepared as flattened whole mounts and stimulated in Ringer buffer with or without 59 mM KCl. To investigate VNUT expression, sections of the mouse retinas were evaluated with immunohistochemistry and western blot analysis using newly developed antibodies against VNUT. All images were examined and photographed under confocal microscopy. Electroretinogram (ERG) recordings were performed on the C57BL/6J and DBA/2J mice to analyze the changes in the electrophysiological response; a decrease in the scotopic threshold response was observed in the 15-month-old DBA/2J mice. Results In the 15-month-old control and glaucomatous mice, electrophysiological changes of 42% were observed. In addition, 50% increases in the intraocular pressure (IOP) were observed when the pathology was fully established. The responses in the retinal ATP net release as the pathology progressed varied from 0.32±0.04 pmol/retina (3 months) to 1.10±0.06 pmol/retina (15 months; threefold increase). Concomitantly, VNUT expression was significantly increased during glaucoma progression in the DBA/2J mice (58%) according to the immunohistochemical and western blot analysis. Conclusions These results may indicate a possible correlation between retinal dysfunction and increased levels of extracellular ATP and nucleotide transporter. These data support an excitotoxicity role for ATP via P2X7R in glaucoma. This modified cellular environment could contribute to explaining the functional and biochemical alterations observed during the development of the pathology. PMID:26392744

  11. Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

    PubMed Central

    Charoonroje, P; Tokumitsu, Y; Nomura, Y

    1994-01-01

    1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system. PMID:7834209

  12. Increased astrocytic ATP release results in enhanced excitability of the hippocampus.

    PubMed

    Lee, Hae Ung; Yamazaki, Yoshihiko; Tanaka, Kenji F; Furuya, Kishio; Sokabe, Masahiro; Hida, Hideki; Takao, Keizo; Miyakawa, Tsuyoshi; Fujii, Satoshi; Ikenaka, Kazuhiro

    2013-02-01

    Astrocytes, a major subtype of glia, interact with neurons as a supportive partner supplying energy sources and growth factors. Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters (gliotransmitters). However, the precise role of gilotransmitters in regulating neuronal activity is still under debate. Here, we report that a subtle enhancement in the release of one gliotransmitter, ATP, affects synaptic potentiation from an analysis of mice containing an astrocyte-selective (GFAP) mutation. We found that, relative to normal mice, weaker stimulation induced long-term potentiation (LTP) in mutant mice, indicating that the threshold to induce LTP was lowered in the mutant. While excitatory transmission was normal in the mutant, inhibitory GABAergic transmission was suppressed. We found that a low concentration of adenosine selectively attenuated inhibitory neuronal activity and lowered the threshold to induce LTP in wild type mice. In comparison, adenosine A(1) receptor antagonism reversed the lowered LTP threshold back to normal in the mutant mouse. We verified that adenosine levels in the cerebrospinal fluid of mutant mice were slightly elevated compared to wild type mice. This was apparently caused by an increase in ATP release from mutant astrocytes that could provide a source of augmented adenosine levels in the mutant. ATP is thought to suppress the excitability of neuronal circuits; however, a small increase in ATP release can result in a suppressed inhibitory tone and enhanced excitability of neuronal circuitry. These findings demonstrate that ATP released from astrocytes acts in a bidirectional fashion to regulate neuronal excitability depending on concentration. PMID:23018918

  13. Magnesium inhibits the calcification of the extracellular matrix in tendon-derived stem cells via the ATP-P2R and mitochondrial pathways.

    PubMed

    Yue, Jiaji; Jin, Shanzi; Li, Yaqiang; Zhang, Li; Jiang, Wenwei; Yang, Chunxi; Du, Jiang

    2016-09-01

    Tendon calcification has been widely regarded by researchers to result from the osteogenic differentiation of Tendon-Derived Stem Cells (TDSCs) and ectopic mineralization caused by the calcification of cellular matrix. Recent studies have revealed a correlation between the Mg(2+)/Ca(2+) balance and the degeneration or calcification of tendon tissues. Furthermore, the ATP-P2X/P2Y receptor pathway has been shown to play a decisive role in the process of calcification, with calcium exportation from mitochondria and calcium oscillations potentially representing the cohesive signal produced by this pathway. Our previous study demonstrated that matrix calcification is inhibited by magnesium. In this study, we examined the effects of extracellular Mg(2+) on the deposition of calcium phosphate matrix and cellular pathways in TDSCs. The suppression of the export of calcium from mitochondria has also been detected. We found that a high concentration of extracellular Mg(2+) ([Mg(2+)]e) inhibited the mineralization of the extracellular matrix in TDSCs and that 100 μM ATP reversed this inhibitory effect in vitro. In addition, the spontaneous release of ATP was inhibited by high [Mg(2+)]e levels. A high [Mg(2+)]e suppressed the expression of P2X4, P2X5 and P2X7 and activated the expression of P2Y1, P2Y2, P2Y4 and P2Y14. The interaction between Mg(2+) and Ca(2+) is therefore contradictory, Mg(2+) inhibits mitochondrial calcium concentrations, meanwhile it reverses the opening of mPTP that is induced by Ca(2+). JC-1 staining verified the protective effect of Mg(2+) on mitochondrial membrane potential and the decrease induced by Ca(2+). Taken together, these results indicate that high [Mg(2+)]e interferes with the expression of P2 receptors, resulting in decreased extracellular mineralization. The balance between Mg(2+) and Ca(2+) influences mitochondrial calcium exportation and provides another explanation for the mechanism underlying matrix calcification in TDSCs. PMID

  14. Efficient production and enhanced tumor delivery of engineered extracellular vesicles.

    PubMed

    Watson, Dionysios C; Bayik, Defne; Srivatsan, Avinash; Bergamaschi, Cristina; Valentin, Antonio; Niu, Gang; Bear, Jenifer; Monninger, Mitchell; Sun, Mei; Morales-Kastresana, Aizea; Jones, Jennifer C; Felber, Barbara K; Chen, Xiaoyuan; Gursel, Ihsan; Pavlakis, George N

    2016-10-01

    Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded ∼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods. PMID:27522254

  15. Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force

    PubMed Central

    Glasser, Nathaniel R.; Kern, Suzanne E.

    2014-01-01

    Summary While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton-motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox-active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation. PMID:24612454

  16. Effects of chemical inhibitors and apyrase enzyme further document a role for apyrases and extracellular ATP in the opening and closing of stomates in Arabidopsis

    PubMed Central

    Clark, Greg; Darwin, Cameron; Mehta, Viraj; Jackobs, Faith; Perry, Tyler; Hougaard, Katia; Roux, Stan

    2013-01-01

    In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of apyrase chemical inhibitors can induce stomatal opening in the dark, while apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results further confirm the role of extracellular ATP in regulating stomatal apertures. PMID:23989340

  17. Bioenergetic analysis of intact mammalian cells using the Seahorse XF24 Extracellular Flux analyzer and a luciferase ATP assay.

    PubMed

    de Moura, Michelle Barbi; Van Houten, Bennett

    2014-01-01

    Metabolic pathways and bioenergetics were described in great detail over half a century ago, and during the past decade there has been a resurgence in integrating these cellular processes with other biological properties of the cell, including growth control, protein kinase cascade signaling, cell cycle division, and autophagy. Since many disease conditions are associated with altered metabolism and production of energy, it is important to develop new approaches to measure these cellular parameters. This chapter summarizes a new and exciting approach based on the Seahorse XF24 Extracelluar Flux analyzer, which takes real time measurements of oxidative phosphorylation and glycolysis in living cells. These bioenergetic profiles are then compared with steady-state levels of cellular ATP as measured by a luciferase assay.

  18. A facile label-free aptasensor for detecting ATP based on fluorescence enhancement of poly(thymine)-templated copper nanoparticles.

    PubMed

    Zhou, Sai-Sai; Zhang, Lin; Cai, Qi-Yong; Dong, Zhen-Zhen; Geng, Xin; Ge, Jia; Li, Zhao-Hui

    2016-09-01

    A label-free fluorescence assay has been developed for sensitive and selective detection of adenosine triphosphate (ATP) by using poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) as fluorescent indicator. In our design, ATP aptamer was split into two fragments, both of which were elongated with poly T strands that can be utilized as efficient template for the formation of copper nanoparticles through the reduction of copper ions by sodium ascorbate. In the presence of ATP, the two split aptamers could be dragged to form aptamer-ATP aptamer complex, which drew the poly T strands close to each other and induced a remarkable fluorescence enhancement of poly T-templated CuNPs. Thus, an elevated fluorescence enhancement of poly T-templated CuNPs was obtained with the increase in ATP concentration. Under optimized conditions, a good linear range for ATP detection was realized from 100 nM to 100 μM with a detection limit of 10.29 nM. In addition, the application of this biosensing system in complex biological matrix was demonstrated with satisfactory results. This assay provided a simple, label-free, cost-effective, and sensitive platform for the detection of ATP. PMID:27457102

  19. A facile label-free aptasensor for detecting ATP based on fluorescence enhancement of poly(thymine)-templated copper nanoparticles.

    PubMed

    Zhou, Sai-Sai; Zhang, Lin; Cai, Qi-Yong; Dong, Zhen-Zhen; Geng, Xin; Ge, Jia; Li, Zhao-Hui

    2016-09-01

    A label-free fluorescence assay has been developed for sensitive and selective detection of adenosine triphosphate (ATP) by using poly(thymine) (poly T)-templated copper nanoparticles (CuNPs) as fluorescent indicator. In our design, ATP aptamer was split into two fragments, both of which were elongated with poly T strands that can be utilized as efficient template for the formation of copper nanoparticles through the reduction of copper ions by sodium ascorbate. In the presence of ATP, the two split aptamers could be dragged to form aptamer-ATP aptamer complex, which drew the poly T strands close to each other and induced a remarkable fluorescence enhancement of poly T-templated CuNPs. Thus, an elevated fluorescence enhancement of poly T-templated CuNPs was obtained with the increase in ATP concentration. Under optimized conditions, a good linear range for ATP detection was realized from 100 nM to 100 μM with a detection limit of 10.29 nM. In addition, the application of this biosensing system in complex biological matrix was demonstrated with satisfactory results. This assay provided a simple, label-free, cost-effective, and sensitive platform for the detection of ATP.

  20. The pathophysiology of extracellular hemoglobin associated with enhanced oxidative reactions

    PubMed Central

    Rifkind, Joseph M.; Mohanty, Joy G.; Nagababu, Enika

    2015-01-01

    Hemoglobin (Hb) continuously undergoes autoxidation producing superoxide which dismutates into hydrogen peroxide (H2O2) and is a potential source for subsequent oxidative reactions. Autoxidation is most pronounced under hypoxic conditions in the microcirculation and for unstable dimers formed at reduced Hb concentrations. In the red blood cell (RBC), oxidative reactions are inhibited by an extensive antioxidant system. For extracellular Hb, whether from hemolysis of RBCs and/or the infusion of Hb-based blood substitutes, the oxidative reactions are not completely neutralized by the available antioxidant system. Un-neutralized H2O2 oxidizes ferrous and ferric Hbs to Fe(IV)-ferrylHb and OxyferrylHb, respectively. FerrylHb further reacts with H2O2 producing heme degradation products and free iron. OxyferrylHb, in addition to Fe(IV) contains a free radical that can undergo additional oxidative reactions. Fe(III)Hb produced during Hb autoxidation also readily releases heme, an additional source for oxidative stress. These oxidation products are a potential source for oxidative reactions in the plasma, but to a greater extent when the lower molecular weight Hb dimers are taken up into cells and tissues. Heme and oxyferryl have been shown to have a proinflammatory effect further increasing their potential for oxidative stress. These oxidative reactions contribute to a number of pathological situations including atherosclerosis, kidney malfunction, sickle cell disease, and malaria. The toxic effects of extracellular Hb are of particular concern with hemolytic anemia where there is an increase in hemolysis. Hemolysis is further exacerbated in various diseases and their treatments. Blood transfusions are required whenever there is an appreciable decrease in RBCs due to hemolysis or blood loss. It is, therefore, essential that the transfused blood, whether stored RBCs or the blood obtained by an Autologous Blood Recovery System from the patient, do not further increase

  1. Surface-Enhanced Raman Spectroscopy Study of 4-ATP on Gold Nanoparticles for Basal Cell Carcinoma Fingerprint Detection

    NASA Astrophysics Data System (ADS)

    Quynh, Luu Manh; Nam, Nguyen Hoang; Kong, K.; Nhung, Nguyen Thi; Notingher, I.; Henini, M.; Luong, Nguyen Hoang

    2016-05-01

    The surface-enhanced Raman signals of 4-aminothiophenol (4-ATP) attached to the surface of colloidal gold nanoparticles with size distribution of 2 to 5 nm were used as a labeling agent to detect basal cell carcinoma (BCC) of the skin. The enhanced Raman band at 1075 cm-1 corresponding to the C-S stretching vibration in 4-ATP was observed during attachment to the surface of the gold nanoparticles. The frequency and intensity of this band did not change when the colloids were conjugated with BerEP4 antibody, which specifically binds to BCC. We show the feasibility of imaging BCC by surface-enhanced Raman spectroscopy, scanning the 1075 cm-1 band to detect the distribution of 4-ATP-coated gold nanoparticles attached to skin tissue ex vivo.

  2. V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption

    PubMed Central

    Yang, De-Qin; Feng, Shengmei; Chen, Wei; Zhao, Haibo; Paulson, Christie; Li, Yi-Ping

    2014-01-01

    Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet, the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H+-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of ERK phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and Tm7sf4 expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small GTPase Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease. PMID:22467241

  3. ATP regulation in bioproduction.

    PubMed

    Hara, Kiyotaka Y; Kondo, Akihiko

    2015-12-10

    Adenosine-5'-triphosphate (ATP) is consumed as a biological energy source by many intracellular reactions. Thus, the intracellular ATP supply is required to maintain cellular homeostasis. The dependence on the intracellular ATP supply is a critical factor in bioproduction by cell factories. Recent studies have shown that changing the ATP supply is critical for improving product yields. In this review, we summarize the recent challenges faced by researchers engaged in the development of engineered cell factories, including the maintenance of a large ATP supply and the production of cell factories. The strategies used to enhance ATP supply are categorized as follows: addition of energy substrates, controlling pH, metabolic engineering of ATP-generating or ATP-consuming pathways, and controlling reactions of the respiratory chain. An enhanced ATP supply generated using these strategies improves target production through increases in resource uptake, cell growth, biosynthesis, export of products, and tolerance to toxic compounds.

  4. Enhanced Invasion of Metastatic Cancer Cells via Extracellular Matrix Interface

    PubMed Central

    Zhu, Jiangrui; Liang, Long; Jiao, Yang; Liu, Liyu

    2015-01-01

    Cancer cell invasion is a major component of metastasis and is responsible for extensive cell diffusion into and major destruction of tissues. Cells exhibit complex invasion modes, including a variety of collective behaviors. This phenomenon results in the structural heterogeneity of the extracellular matrix (ECM) in tissues. Here, we systematically investigated the environmental heterogeneity facilitating tumor cell invasion via a combination of in vitro cell migration experiments and computer simulations. Specifically, we constructed an ECM microenvironment in a microfabricated biochip and successfully created a three-dimensional (3D) funnel-like matrigel interface inside. Scanning electron microscopy demonstrated that the interface was at the interior defects of the nano-scale molecular anisotropic orientation and the localized structural density variations in the matrigel. Our results, particularly the correlation of the collective migration pattern with the geometric features of the funnel-like interface, indicate that this heterogeneous in vitro ECM structure strongly guides and promotes aggressive cell invasion in the rigid matrigel space. A cellular automaton model was proposed based on our experimental observations, and the associated quantitative analysis indicated that cell invasion was initiated and controlled by several mechanisms, including microenvironment heterogeneity, long-range cell-cell homotype and gradient-driven directional cellular migration. Our work shows the feasibility of constructing a complex and heterogeneous in vitro 3D ECM microenvironment that mimics the in vivo environment. Moreover, our results indicate that ECM heterogeneity is essential in controlling collective cell invasive behaviors and therefore determining metastasis efficiency. PMID:25706718

  5. Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

    PubMed

    Kennedy, H J; Pouli, A E; Ainscow, E K; Jouaville, L S; Rizzuto, R; Rutter, G A

    1999-05-01

    Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary beta-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.

  6. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells

    PubMed Central

    Kim, Jayoung; Morley, Samantha; Le, Minh; Bedoret, Denis; Umetsu, Dale T; Di Vizio, Dolores; Freeman, Michael R

    2014-01-01

    The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and DIAPH3 silencing evokes a transition to an amoeboid tumor phenotype in multiple cell backgrounds. This amoeboid transformation is accompanied by increased tumor cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the formation of atypically large (>1 μm) membrane blebs that can be shed as extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of nano-sized EV (e.g., exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused EV shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of EV production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells. DU145 EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV produced as a result of DIAPH3 loss or growth factor stimulation may condition the tumor microenvironment through multiple mechanisms, including the proliferation of cancer cells and suppression of tumor-infiltrating immune cells. PMID:24423651

  7. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  8. A Common Molecular Motif Characterizes Extracellular Allosteric Enhancers of GPCR Aminergic Receptors and Suggests Enhancer Mechanism of Action

    PubMed Central

    Bernstein, Robert Root; Dillon, Patrick F

    2014-01-01

    Several classes of compounds that have no intrinsic activity on aminergic systems nonetheless enhance the potency of aminergic receptor ligands three-fold or more while significantly increasing their duration of activity, preventing tachyphylaxis and reversing fade. Enhancer compounds include ascorbic acid, ethylenediaminetetraacetic acid, cortico-steroids, opioid peptides, opiates and opiate antagonists. This paper provides the first review of aminergic enhancement, demonstrating that all enhancers have a common, inobvious molecular motif and work through a common mechanism that is manifested by three common characteristics. First, aminergic enhancers bind directly to the amines they enhance, suggesting that the common structural motif is reflected in common binding targets. Second, one common target is the first extracellular loop of aminergic receptors. Third, at least some enhancers are antiphosphodiesterases. These observations suggest that aminergic enhancers act on the extracellular surface of aminergic receptors to keep the receptor in its high affinity state, trapping the ligand inside the receptor. Enhancer binding produces allosteric modifications of the receptor structure that interfere with phosphorylation of the receptor, thereby inhibiting down-regulation of the receptor. The mechanism explains how enhancers potentiate aminergic activity and increase duration of activity and makes testable predictions about additional compounds that should act as aminergic enhancers. PMID:25174918

  9. Current data on ATP-containing liposomes and potential prospects to enhance cellular energy status for hepatic applications.

    PubMed

    Korb, V; Tep, K; Escriou, V; Richard, C; Scherman, D; Cynober, L; Chaumeil, J; Dumortier, G

    2008-01-01

    The pharmacological use of adenosine triphosphate (ATP), although promising, is restricted due to poor cellular penetration and drastic hydrolysis that is markedly accelerated in vivo by ectoenzymes. In the literature, liposomes have proven efficient in offering a physical barrier to extracellular enzymes and favor penetration into cells. First, this review addresses the issues raised by ATP development in pharmaceutics. Second, studies conducted with ATP liposomally entrapped (lipo-ATP) are described, including pharmaco-technical formulation engineering and related models of assessment. Finally, potential directions for research to better target ATP penetration into the liver are considered. Lipo-ATP were formulated for a number of applications, including sepsis-related disorders; spermatozoid alteration; brain ischemia episodes; and ophthalmic, cardiac, and hepatic use. Key formulation parameters need to be carefully considered to optimize stability and entrapment yield value, and to define the manufacturing process. Positive lipids, such as stearylamine, increase entrapment yield value by electrostatic interaction with negatively charged ATP. A freezing-thawing step in the manufacturing process considerably increases entrapment yield value. Lipo-ATP were assessed using cell culture, isolated organs, and animal experimental models. Very promising results were obtained with antimyosin PEGylated immunoliposomes using isolated rat hearts and experimental myocardial infarction in rabbits. In hepatic applications, lipo-ATP are effective in preventing liver injury during shock and to improve the energy status of cold-stored rat liver, in particular, if liposomes are loaded with apolipoprotein E (ApoE). For liver delivery, liposome size needs to be lower than 100 nm to allow diffusion through the Disse space, but liposome flexibility and lipid content may also influence liver uptake. The role of the liposome charge remains unclear. ApoE and the ligand for the

  10. Enhancement of ATP generation capacity, antioxidant activity and immunomodulatory activities by Chinese Yang and Yin tonifying herbs

    PubMed Central

    Ko, Kam Ming; Leung, Hoi Yan

    2007-01-01

    Chinese tonifying herbs such as Herba Cistanche, Ganoderma and Cordyceps, which possess antioxidant and/or immunomodulatory activities, can be useful in the prevention and treatment of age-related diseases. Pharmacological studies on Yang and Yin tonifying herbs suggest that Yang tonifying herbs stimulate mitochondrial adenosine triphosphate (ATP) generation, presumably through the intermediacy of reactive oxidant species, leading to the enhancement of cellular/mitochondrial antioxidant status. Yin tonifying herbs, however, apart from possessing antioxidant properties, exert mainly immunomodulatory functions that may boost a weak immune system and may also suppress overreactive immune responses. The abilities of Yang and Yin Chinese tonifying herbs to enhance ATP generation and to exhibit antioxidant and/or immunomodulatory actions are the pharmacological basis for their beneficial effects on the retardation of aging. PMID:17386115

  11. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    PubMed

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  12. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    PubMed

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.

  13. The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids, mannosylerythritol lipids, in the basidiomycetous yeast Pseudozyma antarctica.

    PubMed

    Morita, Tomotake; Ito, Emi; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

    2010-07-01

    Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface-active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL-producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1-PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. PMID:20146402

  14. Intracellular and Extracellular Carbonic Anhydrases Cooperate Non-enzymatically to Enhance Activity of Monocarboxylate Transporters*

    PubMed Central

    Klier, Michael; Andes, Fabian T.; Deitmer, Joachim W.; Becker, Holger M.

    2014-01-01

    Proton-coupled monocarboxylate transporters (MCTs) are carriers of high-energy metabolites such as lactate, pyruvate, and ketone bodies and are expressed in most tissues. It has previously been shown that transport activity of MCT1 and MCT4 is enhanced by the cytosolic carbonic anhydrase II (CAII) independent of its catalytic activity. We have now studied the influence of the extracellular, membrane-bound CAIV on transport activity of MCT1/4, heterologously expressed in Xenopus oocytes. Coexpression of CAIV with MCT1 and MCT4 resulted in a significant increase in MCT transport activity, even in the nominal absence of CO2/HCO3−. CAIV-mediated augmentation of MCT activity was independent of the CAIV catalytic function, since application of the CA-inhibitor ethoxyzolamide or coexpression of the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT transport activity. The interaction required CAIV at the extracellular surface, since injection of CAIV protein into the oocyte cytosol did not augment MCT transport function. The effects of cytosolic CAII (injected as protein) and extracellular CAIV (expressed) on MCT transport activity, were additive. Our results suggest that intra- and extracellular carbonic anhydrases can work in concert to ensure rapid shuttling of metabolites across the cell membrane. PMID:24338019

  15. ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena.

    PubMed

    Ning, YingZhi; Dang, Huai; Liu, GuangLong; Xiong, Jie; Yuan, DongXia; Feng, LiFang; Miao, Wei

    2015-03-01

    The reuse of dichlorodiphenyltrichloroethane (DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down (abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L(-1) DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.

  16. Purinergic Signaling in Early Inflammatory Events of the Foreign Body Response: Modulating Extracellular ATP as an Enabling Technology for Engineered Implants and Tissues

    PubMed Central

    Fann, Stephen A.; Yost, Michael J.

    2014-01-01

    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. PMID:24279914

  17. On the mechanism of enhanced ATP formation in hypoxic myocardium caused by glutamic acid.

    PubMed

    Pisarenko, O I; Solomatina, E S; Ivanov, V E; Studneva, I M; Kapelko, V I; Smirnov, V N

    1985-01-01

    The effect of glutamic acid on the cardiac contractile function and sources of anaerobic ATP formation in hypoxic myocardium was studied in isovolumic rat hearts. The presence of glutamic acid (5 mM) in the perfusate significantly diminished an increment in diastolic pressure caused by 60 min hypoxia, and facilitated its complete recovery during 30 min reoxygenation. This effect was combined with the maintenance of a higher ATP level during hypoxia and reoxygenation. The total content of lactate in the heart-perfusate system rose exactly as during hypoxia without glutamic acid, while pyruvate content decreased due to increased alanine formation. Restoration of tissue content of glutamate and aspartate in the presence of exogenous glutamic acid was accompanied by a more than 2-fold increase in succinate formation, the end-product of the Krebs' cycle under anaerobic conditions. The products of glutamic acid transamination with oxaloacetic acid, aspartic and alpha-ketoglutaric acids (5mM each), induced the same functional and metabolic alterations as glutamic acid. Amino-oxyacetic acid, a tramsaminase inhibitor, eliminated the effects caused by glutamic acid. Moreover, the inhibition of transamination was accompanied by a decreased succinate and alanine synthesis as well as insignificantly increased lactate formation compared to hypoxia without additives. The results suggest that the beneficial effect of glutamic acid is due to the activation of anaerobic ATP formation in the mitochondria rather than stimulation of glycolysis.

  18. Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

    PubMed

    Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

    2016-06-01

    Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

  19. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  20. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    PubMed

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production.

  1. Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

    PubMed Central

    Okamoto, Akihiro; Hashimoto, Kazuhito; Nealson, Kenneth H.; Nakamura, Ryuhei

    2013-01-01

    Extracellular redox-active compounds, flavins and other quinones, have been hypothesized to play a major role in the delivery of electrons from cellular metabolic systems to extracellular insoluble substrates by a diffusion-based shuttling two-electron-transfer mechanism. Here we show that flavin molecules secreted by Shewanella oneidensis MR-1 enhance the ability of its outer-membrane c-type cytochromes (OM c-Cyts) to transport electrons as redox cofactors, but not free-form flavins. Whole-cell differential pulse voltammetry revealed that the redox potential of flavin was reversibly shifted more than 100 mV in a positive direction, in good agreement with increasing microbial current generation. Importantly, this flavin/OM c-Cyts interaction was found to facilitate a one-electron redox reaction via a semiquinone, resulting in a 103- to 105-fold faster reaction rate than that of free flavin. These results are not consistent with previously proposed redox-shuttling mechanisms but suggest that the flavin/OM c-Cyts interaction regulates the extent of extracellular electron transport coupled with intracellular metabolic activity. PMID:23576738

  2. Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant.

    PubMed

    Lacroix, Benoît; Citovsky, Vitaly

    2011-01-01

    VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell. PMID:22028781

  3. A label-free aptasensor for highly sensitive detection of ATP and thrombin based on metal-enhanced PicoGreen fluorescence.

    PubMed

    Wang, Kaiyu; Liao, Jian; Yang, Xiangyue; Zhao, Meng; Chen, Min; Yao, Weirong; Tan, Weihong; Lan, Xiaopeng

    2015-01-15

    A label-free fluorescence aptasensor for highly selective and sensitive detection of ATP and thrombin was developed by using PicoGreen (PG) as signal molecule and surface-bound metal-enhanced fluorescence (MEF) substrates (silver island films, SIFs) as signal enhancers. On binding with ATP or thrombin, aptamers undergo structure switching, leading to a reduction of fluorescence intensity of PG. Chang of fluorescence intensity can be magnified by SIFs. The limit of detection for ATP and thrombin is 1.3 nM and 0.073 nM, respectively. The fluorescence quenching efficiency is linear in the logarithmic scale with ATP concentration range from 10 nM to 100 μM (R(2)=0.995) and thrombin concentration range from 0.1 nM to 100 nM (R(2)=0.997). The coefficients of variation of the intra-assay reproducibility and inter-assay reproducibility for ATP (10 μM) assay are 3.8% and 5.2%, respectively. In addition, the aptasensor is stable and can be reliably used for ATP measurement in biological samples. Overall, the aptasensor can be a useful and cost effective tool for the specific detection of ATP, thrombin and potentially other biomolecules in biological samples. PMID:25086329

  4. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  5. Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species

    PubMed Central

    Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

    2016-01-01

    The successful treatment of bacterial infections is the achievement of a synergy between the host’s immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host’s immune defences and antibiotic interactions in microbial infections. PMID:26778774

  6. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis

    PubMed Central

    Nakase, Ikuhiko; Noguchi, Kosuke; Fujii, Ikuo; Futaki, Shiroh

    2016-01-01

    Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained. PMID:27748399

  7. Nitrogen doped carbon nanoparticles enhanced extracellular electron transfer for high-performance microbial fuel cells anode.

    PubMed

    Yu, Yang-Yang; Guo, Chun Xian; Yong, Yang-Chun; Li, Chang Ming; Song, Hao

    2015-12-01

    Nitrogen doped carbon nanoparticles (NDCN) were applied to modify the carbon cloth anodes of microbial fuel cells (MFCs) inoculated with Shewanella oneidensis MR-1, one of the most well-studied exoelectrogens. Experimental results demonstrated that the use of NDCN increased anodic absorption of flavins (i.e., the soluble electron mediator secreted by S. oneidensis MR-1), facilitating shuttle-mediated extracellular electron transfer. In addition, we also found that NDCN enabled enhanced contact-based direct electron transfer via outer-membrane c-type cytochromes. Taken together, the performance of MFCs with the NDCN-modified anode was enormously enhanced, delivering a maximum power density 3.5 times' higher than that of the MFCs without the modification of carbon cloth anodes.

  8. Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.

    PubMed

    Hunsberger, Holly C; Wang, Desheng; Petrisko, Tiffany J; Alhowail, Ahmad; Setti, Sharay E; Suppiramaniam, Vishnu; Konat, Gregory W; Reed, Miranda N

    2016-07-01

    Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24 h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of

  9. ACTIVATION OF EXTRACELLULAR-SIGNAL REGULATED KINASE (ERK1/2) BY FLUID SHEAR IS CA2+- AND ATP-DEPENDENT IN MC3T3-E1 OSTEOBLASTS

    PubMed Central

    Liu, Dawei; Genetos, Damian C.; Shao, Ying; Geist, Derik J.; Li, Jiliang; Ke, Hua Zhu; Turner, Charles H.; Duncan, Randall L.

    2010-01-01

    To determine the role of Ca2+ signaling in activation of the Mitogen-Activated Protein Kinase (MAPK) pathway, we subjected MC3T3-E1 pre-osteoblastic cells to inhibitors of Ca2+ signaling during application of fluid shear stress (FSS). FSS only activated ERK1/2, rapidly inducing phosphorylation within 5 minutes of the onset of shear. Phosphorylation of ERK1/2 (pERK1/2) was significantly reduced when Ca2+i was chelated with BAPTA or when Ca2+ was removed from the flow media. Inhibition of both the L-type voltage-sensitive Ca2+ channel and the mechanosensitive cation-selective channel blocked FSS-induced pERK1/2. Inhibition of phospholipase C with U73122 significantly reduced pERK1/2. This inhibition did not result from block of intracellular Ca2+ release, but a loss of PKC activation. Recent data suggests a role of ATP release and purinergic receptor activation in mechanotransduction. Apyrase-mediated hydrolysis of extracellular ATP completely blocked FSS-induced phosphorylation of ERK1/2, while addition of exogenous ATP to static cells mimicked the effects of FSS on pERK1/2. Two P2 receptors, P2Y2 and P2X7, have been associated with the anabolic responses of bone to mechanical loading. Using both iRNA techniques and primary osteoblasts isolated from P2X7 knockout mice, we found that the P2X7, but not the P2Y2, purinergic receptor was involved in ERK1/2 activation under FSS. These data suggest that FSS-induced ERK1/2 phosphorylation requires Ca2+-dependent ATP release, however both increased Ca2+i and PKC activation are needed for complete activation. Further, this ATP-dependent ERK1/2 phosphorylation is mediated through P2X7, but not P2Y2, purinergic receptors. PMID:18291742

  10. Roles of extracellular polymeric substances in enhanced biological phosphorus removal process.

    PubMed

    Li, Wen-Wei; Zhang, Hai-Ling; Sheng, Guo-Ping; Yu, Han-Qing

    2015-12-01

    Enhanced biological phosphorus removal (EBPR) process is known to mainly rely on the ability of phosphorus-accumulating organisms to take up, transform and store excess amount of phosphorus (P) inside the cells. However, recent studies have revealed considerable accumulation of P also in the extracellular polymeric substances (EPS) of sludge, implying a non-negligible role of EPS in P removal by EBPR sludge. However, the contribution of EPS to P uptake and the forms of accumulated extracellular P vary substantially in different studies, and the underlying mechanism of P transformation and transportation in EPS remains poorly understood. This review provides a new recognition into the P removal process in EBPR system by incorporating the role of EPS. It overviews on the characteristics of P accumulation in EPS, explores the mechanism of P transformation and transportation in EBPR sludge and EPS, summarizes the main influential factors for the P-accumulation properties of EPS, and discusses the remaining knowledge gaps and needed future efforts that may lead to better understanding and use of such an EPS role for maximizing P recovery from wastewater. PMID:26143588

  11. Roles of extracellular polymeric substances in enhanced biological phosphorus removal process.

    PubMed

    Li, Wen-Wei; Zhang, Hai-Ling; Sheng, Guo-Ping; Yu, Han-Qing

    2015-12-01

    Enhanced biological phosphorus removal (EBPR) process is known to mainly rely on the ability of phosphorus-accumulating organisms to take up, transform and store excess amount of phosphorus (P) inside the cells. However, recent studies have revealed considerable accumulation of P also in the extracellular polymeric substances (EPS) of sludge, implying a non-negligible role of EPS in P removal by EBPR sludge. However, the contribution of EPS to P uptake and the forms of accumulated extracellular P vary substantially in different studies, and the underlying mechanism of P transformation and transportation in EPS remains poorly understood. This review provides a new recognition into the P removal process in EBPR system by incorporating the role of EPS. It overviews on the characteristics of P accumulation in EPS, explores the mechanism of P transformation and transportation in EBPR sludge and EPS, summarizes the main influential factors for the P-accumulation properties of EPS, and discusses the remaining knowledge gaps and needed future efforts that may lead to better understanding and use of such an EPS role for maximizing P recovery from wastewater.

  12. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    PubMed

    Sours, Kevin M; Xiao, Yao; Ahn, Natalie G

    2014-05-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.

  13. 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.

  14. A thermonuclease of Neisseria gonorrhoeae enhances bacterial escape from killing by neutrophil extracellular traps.

    PubMed

    Juneau, Richard A; Stevens, Jacqueline S; Apicella, Michael A; Criss, Alison K

    2015-07-15

    Acute gonorrhea is characterized by neutrophilic inflammation that is insufficient to clear Neisseria gonorrhoeae. Activated neutrophils release extracellular traps (NETs), which are composed of chromatin and decorated with antimicrobial proteins. The N. gonorrhoeae NG0969 open reading frame contains a gene (nuc) that encodes a putatively secreted thermonuclease (Nuc) that contributes to biofilm remodeling. Here, we report that Nuc degrades NETs to help N. gonorrhoeae resist killing by neutrophils. Primary human neutrophils released NETs after exposure to N. gonorrhoeae, but NET integrity declined over time with Nuc-containing bacteria. Recombinant Nuc and conditioned medium from Nuc-containing N. gonorrhoeae degraded human neutrophil DNA and NETs. NETs were found to have antimicrobial activity against N. gonorrhoeae, and Nuc expression enhanced N. gonorrhoeae survival in the presence of neutrophils that released NETs. We propose that Nuc enables N. gonorrhoeae to escape trapping and killing by NETs during symptomatic infection, highlighting Nuc as a multifunctional virulence factor for N. gonorrhoeae.

  15. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

    PubMed

    Clough, Bret H; McCarley, Matthew R; Krause, Ulf; Zeitouni, Suzanne; Froese, Jeremiah J; McNeill, Eoin P; Chaput, Christopher D; Sampson, H Wayne; Gregory, Carl A

    2015-01-01

    Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

  16. 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. PMID:27088570

  17. Enhancement of rat bladder contraction by artificial sweeteners via increased extracellular Ca{sup 2+} influx

    SciTech Connect

    Dasgupta, Jaydip; Elliott, Ruth A. . E-mail: rae5@leicester.ac.uk; Doshani, Angie; Tincello, Douglas G.

    2006-12-01

    Introduction: Consumption of carbonated soft drinks has been shown to be independently associated with the development of overactive bladder symptoms (OR 1.62, 95% CI 1.18, 2.22) [Dallosso, H.M., McGrother, C.W., Matthews, R.J., Donaldson, M.M.K., 2003. The association of diet and other lifestyle factors with overactive bladder and stress incontinence: a longitudinal study in women. BJU Int. 92, 69-77]. We evaluated the effects of three artificial sweeteners, acesulfame K, aspartame and sodium saccharin, on the contractile response of isolated rat detrusor muscle strips. Methods: Strips of detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with {alpha},{beta} methylene ATP, potassium, calcium and carbachol. Results: Sweeteners 10{sup -7} M to 10{sup -2} M enhanced the contractile response to 10 Hz EFS compared to control (p < 0.01). The atropine-resistant response to EFS was marginally increased by acesulfame K 10{sup -6} M, aspartame 10{sup -7} M and sodium saccharin 10{sup -7} M. Acesulfame K 10{sup -6} M increased the maximum contractile response to {alpha},{beta} methylene ATP by 35% ({+-} 9.6%) (p < 0.05) and to KCl by 12% ({+-} 3.1%) (p < 0.01). Sodium saccharin also increased the response to KCl by 37% ({+-} 15.2%) (p < 0.05). These sweeteners shifted the calcium concentration-response curves to the left. Acesulfame K 10{sup -6} M increased the log EC{sub 5} from -2.79 ({+-} 0.037) to -3.03 ({+-} 0.048, p < 0.01) and sodium saccharin 10{sup -7} M from -2.74 ({+-} 0.03) to 2.86 ({+-} 0.031, p < 0.05). The sweeteners had no significant effect on the contractile response to carbachol but they did increase the amplitude of spontaneous bladder contractions. Discussion: These results suggest that low concentrations of artificial sweeteners enhanced detrusor muscle contraction via modulation of L-type Ca{sup +2} channels.

  18. The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

    PubMed Central

    Lee, Mark J.; Liu, Hong; Barker, Bridget M.; Snarr, Brendan D.; Gravelat, Fabrice N.; Al Abdallah, Qusai; Gavino, Christina; Baistrocchi, Shane R.; Ostapska, Hanna; Xiao, Tianli; Ralph, Benjamin; Solis, Norma V.; Lehoux, Mélanie; Baptista, Stefanie D.; Thammahong, Arsa; Cerone, Robert P.; Kaminskyj, Susan G. W.; Guiot, Marie-Christine; Latgé, Jean-Paul; Fontaine, Thierry; Vinh, Donald C.; Filler, Scott G.; Sheppard, Donald C.

    2015-01-01

    Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs. PMID:26492565

  19. Enhancement of sludge reduction and methane production by removing extracellular polymeric substances from waste activated sludge.

    PubMed

    Nguyen, Minh Tuan; Mohd Yasin, Nazlina Haiza; Miyazaki, Toshiki; Maeda, Toshinari

    2014-12-01

    The management of waste activated sludge (WAS) recycling is a concern that affects the development of the future low-carbon society, particularly sludge reduction and biomass utilization. In this study, we investigated the effect of removing extracellular polymeric substances (EPS), which play important roles in the adhesion and flocculation of WAS, on increased sludge disintegration, thereby enhancing sludge reduction and methane production by anaerobic digestion. EPS removal from WAS by ethylenediaminetetraacetic acid (EDTA) significantly enhanced sludge reduction, i.e., 49 ± 5% compared with 27 ± 1% of the control at the end the digestion process. Methane production was also improved in WAS without EPS by 8881 ± 109 CH4 μmol g(-1) dry-weight of sludge. Microbial activity was determined by denaturing gradient gel electrophoresis and real-time polymerase chain reaction, which showed that the hydrolysis and acetogenesis stages were enhanced by pretreatment with 2% EDTA, with a larger methanogenic community and better methane production. PMID:25277968

  20. The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination.

    PubMed

    Vono, Maria; Taccone, Marianna; Caccin, Paola; Gallotta, Marilena; Donvito, Giovanna; Falzoni, Simonetta; Palmieri, Emiliano; Pallaoro, Michele; Rappuoli, Rino; Di Virgilio, Francesco; De Gregorio, Ennio; Montecucco, Cesare; Seubert, Anja

    2013-12-24

    Vaccines are the most effective agents to control infections. In addition to the pathogen antigens, vaccines contain adjuvants that are used to enhance protective immune responses. However, the molecular mechanism of action of most adjuvants is ill-known, and a better understanding of adjuvanticity is needed to develop improved adjuvants based on molecular targets that further enhance vaccine efficacy. This is particularly important for tuberculosis, malaria, AIDS, and other diseases for which protective vaccines do not exist. Release of endogenous danger signals has been linked to adjuvanticity; however, the role of extracellular ATP during vaccination has never been explored. Here, we tested whether ATP release is involved in the immune boosting effect of four common adjuvants: aluminum hydroxide, calcium phosphate, incomplete Freund's adjuvant, and the oil-in-water emulsion MF59. We found that intramuscular injection is always associated with a weak transient release of ATP, which was greatly enhanced by the presence of MF59 but not by all other adjuvants tested. Local injection of apyrase, an ATP-hydrolyzing enzyme, inhibited cell recruitment in the muscle induced by MF59 but not by alum or incomplete Freund's adjuvant. In addition, apyrase strongly inhibited influenza-specific T-cell responses and hemagglutination inhibition titers in response to an MF59-adjuvanted trivalent influenza vaccine. These data demonstrate that a transient ATP release is required for innate and adaptive immune responses induced by MF59 and link extracellular ATP with an enhanced response to vaccination. PMID:24324152

  1. Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

    PubMed Central

    Cooke, M. J.; Phillips, S. R.; Shah, D. S.H.; Athey, D.; Lakey, J. H.

    2008-01-01

    Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory. PMID:19002844

  2. Enhancement of extracellular electron transfer and bioelectricity output by synthetic porin.

    PubMed

    Yong, Yang-Chun; Yu, Yang-Yang; Yang, Yun; Liu, Jing; Wang, Jing-Yuan; Song, Hao

    2013-02-01

    The microbial fuel cell (MFC), is a promising environmental biotechnology for harvesting electricity energy from organic wastes. However, low bacterial membrane permeability of electron shuttles is a limiting factor that restricts the electron shuttle-mediated extracellular electron transfer (EET) from bacteria to electrodes, thus the electricity power output of MFCs. To this end, we heterologously expressed a porin protein OprF from Pseudomonas aeruginosa PAO1 into Escherichia coli, which dramatically increased its membrane permeability, delivering a much higher current output in MFCs than its parental strain (BL21). We found that the oprF-expression strain showed more efficient EET than its parental strain. More strikingly, the enhanced membrane permeability also rendered the oprF-expression strain an efficient usage of riboflavin as the electron shuttle, whereas its parental strain was incapable of. Our results substantiated that membrane permeability is crucial for the efficient EET, and indicated that the expression of synthetic porins could be an efficient strategy to enhance bioelectricity generation by microorganisms (including electrogenic bacteria) in MFCs.

  3. Dissolved inorganic carbon uptake in Thiomicrospira crunogena XCL-2 is Δp- and ATP-sensitive and enhances RubisCO-mediated carbon fixation.

    PubMed

    Menning, Kristy J; Menon, Balaraj B; Fox, Gordon; Scott, Kathleen M

    2016-03-01

    The gammaproteobacterium Thiomicrospira crunogena XCL-2 is an aerobic sulfur-oxidizing hydrothermal vent chemolithoautotroph that has a CO2 concentrating mechanism (CCM), which generates intracellular dissolved inorganic carbon (DIC) concentrations much higher than extracellular, thereby providing substrate for carbon fixation at sufficient rate. This CCM presumably requires at least one active DIC transporter to generate the elevated intracellular concentrations of DIC measured in this organism. In this study, the half-saturation constant (K CO2) for purified carboxysomal RubisCO was measured (276 ± 18 µM) which was much greater than the K CO2 of whole cells (1.03 µM), highlighting the degree to which the CCM facilitates CO2 fixation under low CO2 conditions. To clarify the bioenergetics powering active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis (DCCD) or proton potential (CCCP). Incubations with each of these inhibitors resulted in diminished intracellular ATP, DIC, and fixed carbon, despite an absence of an inhibitory effect on proton potential in the DCCD-incubated cells. Electron transport complexes NADH dehydrogenase and the bc 1 complex were found to be insensitive to DCCD, suggesting that ATP synthase was the primary target of DCCD. Given the correlation of DIC uptake to the intracellular ATP concentration, the ABC transporter genes were targeted by qRT-PCR, but were not upregulated under low-DIC conditions. As the T. crunogena genome does not include orthologs of any genes encoding known DIC uptake systems, these data suggest that a novel, yet to be identified, ATP- and proton potential-dependent DIC transporter is active in this bacterium. This transporter serves to facilitate growth by T. crunogena and other Thiomicrospiras in the many habitats where they are found.

  4. Enhanced butanol production by increasing NADH and ATP levels in Clostridium beijerinckii NCIMB 8052 by insertional inactivation of Cbei_4110.

    PubMed

    Liu, Jun; Guo, Ting; Wang, Dong; Shen, Xiaoning; Liu, Dong; Niu, Huanqing; Liang, Lei; Ying, Hanjie

    2016-06-01

    Clostridium beijerinckii is identified as a promising Clostridium strain for industrialization of acetone and butanol (AB) fermentation. It has been reported that high reducing power levels are associated with high butanol yield. In this study, we regulated reducing power by blocking NAD(P)H consumption in C. beijerinckii NCIMB 8052. Gene Cbei_4110, encoding NADH-quinone oxidoreductase (nuoG), is a subunit of the electron transport chain complex I. After inactivation of gene Cbei_4110, the generated mutant strain exhibited a remarkable increase in glucose utilization ratio and enhanced butanol production to 9.5 g/L in P2 medium containing 30 g/L of glucose. NAD(P)H and ATP levels were also increased by one to two times and three to five times, respectively. Furthermore, a comparative transcriptome analysis was carried out in order to determine the mechanism involved in the enhanced activity of the Cbei_4110-inactivated mutant strain. This strategy may be extended for making industrial bio-butanol more economically attractive. PMID:26830101

  5. Opening of ATP-sensitive K(+) (KATP) channels enhance hydroxyl radical generation induced by MPP(+) in rat striatum.

    PubMed

    Obata, Toshio; Nakashima, Michiko

    2016-07-15

    The present study examined whether opening of adenosine triphosphate (ATP) sensitive K(+) (KATP) channels can enhance 1-methyl-4-phenylpyridinium (MPP(+))-induced hydroxyl radical (OH) generation in rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5nmol/ml per min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2.3-dihydroxybenzoic acid (DHBA) in the striatum. MPP(+) (5mM) enhanced generation of OH with concomitant increased efflux of dopamine (DA). Cromakalim (100μM), a KATP channel opener, through the microdialysis probe significantly increased both DA efflux and OH formation induced by MPP(+). Another KATP channel opener, nicorandil (1mM), also increased the level DA or DHBA, but these changes were not significant. However, in the presence of glibenclamide (10μM), a KATP channel antagonist, and the increase of MPP(+)-induced DA or DHBA were not observed. Cromakalim (10, 50 and 100μM) increased MPP(+)-induced DHBA formation in a concentration-dependent manner. However, the effects of cromakalim in the presence of glibenclamide were abolished. These results suggest that opening of KATP channels may cause OH generation by MPP(+). PMID:27288802

  6. Performance of Rodent Spermatozoa Over Time Is Enhanced by Increased ATP Concentrations: The Role of Sperm Competition.

    PubMed

    Tourmente, Maximiliano; Villar-Moya, Pilar; Varea-Sánchez, María; Luque-Larena, Juan J; Rial, Eduardo; Roldan, Eduardo R S

    2015-09-01

    Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa.

  7. ATP release through pannexon channels.

    PubMed

    Dahl, Gerhard

    2015-07-01

    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

  8. Galectin-3 Enhances Extracellular Matrix Associations and Wound Healing in Monkey Corneal Epithelium

    PubMed Central

    Fujii, Atsuko; Shearer, Thomas R.; Azuma, Mitsuyoshi

    2016-01-01

    Poor healing of epithelial wounds in cornea is a major clinical problem, leading to persistent epithelial defects and ulceration. The primary cause is poor cell migration over the wound. Carbohydrate-binding protein galectin-3 binds to extracellular matrixes (ECMs) and promotes lamellipodia formation by cross-linking to α3 integrin. Recombinant galectin-3 also facilitates wound healing in the rodent cornea. The purposes of the present experiments were to: (1) establish epithelial wound healing models in monkey corneal explant culture, the models more relevant to human, (2) evaluate the healing effect of galectin-3 in our models, and (3) determine if galectin-3 enhances cell adhesion by interacting with ECMs on corneal surface and their ligand integrins. Monkey corneas with central wounds produced by sodium hydroxide (NaOH) or n-heptanol were incubated with or without recombinant galectin-3. The defected area was stained with sodium fluorescein. Primary isolated corneal epithelial cells from monkey were cultured with or without galectin-3 on plates coated with ECMs or integrins, and the number of adhering cells was counted. Galectin-3 expression in various eye tissues was visualized by immunoblotting. NaOH caused loss of epithelial cells and basement membrane. n-Heptanol removed epithelial cells, but the basement membrane was retained. These corneal defects spontaneously became smaller in a time-dependent manner. Exogenous galectin-3 enhanced wound healing in both NaOH and n-heptanol models. Galectin-3 also enhanced cell adhesion onto the major ECMs found in the basement and Bowman’s membranes and onto integrins. Relatively high levels of galectin-3 were detected in corneal and conjunctival epithelium, but tear fluid contained negligible galactin-3. These results suggested that the enhanced binding of epithelial cells to ECMs and integrins caused by galectin-3 might promote cell migration over wounded corneal surfaces. Since tear fluid contained relatively low

  9. Galectin-3 enhances extracellular matrix associations and wound healing in monkey corneal epithelium.

    PubMed

    Fujii, Atsuko; Shearer, Thomas R; Azuma, Mitsuyoshi

    2015-08-01

    Poor healing of epithelial wounds in cornea is a major clinical problem, leading to persistent epithelial defects and ulceration. The primary cause is poor cell migration over the wound. Carbohydrate-binding protein galectin-3 binds to extracellular matrixes (ECMs) and promotes lamellipodia formation by cross-linking to α3 integrin. Recombinant galectin-3 also facilitates wound healing in the rodent cornea. The purposes of the present experiments were to: (1) establish epithelial wound healing models in monkey corneal explant culture, the models more relevant to human, (2) evaluate the healing effect of galectin-3 in our models, and (3) determine if galectin-3 enhances cell adhesion by interacting with ECMs on corneal surface and their ligand integrins. Monkey corneas with central wounds produced by sodium hydroxide (NaOH) or n-heptanol were incubated with or without recombinant galectin-3. The defected area was stained with sodium fluorescein. Primary isolated corneal epithelial cells from monkey were cultured with or without galectin-3 on plates coated with ECMs or integrins, and the number of adhering cells was counted. Galectin-3 expression in various eye tissues was visualized by immunoblotting. NaOH caused loss of epithelial cells and basement membrane. n-Heptanol removed epithelial cells, but the basement membrane was retained. These corneal defects spontaneously became smaller in a time-dependent manner. Exogenous galectin-3 enhanced wound healing in both NaOH and n-heptanol models. Galectin-3 also enhanced cell adhesion onto the major ECMs found in the basement and Bowman's membranes and onto integrins. Relatively high levels of galectin-3 were detected in corneal and conjunctival epithelium, but tear fluid contained negligible galactin-3. These results suggested that the enhanced binding of epithelial cells to ECMs and integrins caused by galectin-3 might promote cell migration over wounded corneal surfaces. Since tear fluid contained relatively low

  10. Enhancement of Tenogenic Differentiation of Human Adipose Stem Cells by Tendon-Derived Extracellular Matrix

    PubMed Central

    Yang, Guang; Rothrauff, Benjamin B.; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G.; Tuan, Rocky S.

    2014-01-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering. PMID:24044998

  11. A PPARγ AGONIST ENHANCES BACTERIAL CLEARANCE THROUGH NEUTROPHIL EXTRACELLULAR TRAP FORMATION AND IMPROVES SURVIVAL IN SEPSIS.

    PubMed

    Araújo, Cláudia V; Campbell, Clarissa; Gonçalves-de-Albuquerque, Cassiano F; Molinaro, Raphael; Cody, Mark J; Yost, Christian C; Bozza, Patricia T; Zimmerman, Guy A; Weyrich, Andrew S; Castro-Faria-Neto, Hugo C; Silva, Adriana R

    2016-04-01

    Dysregulation of the inflammatory response against infection contributes to mortality in sepsis. Inflammation provides critical host defense, but it can cause tissue damage, multiple organ failure, and death. Because the nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) exhibits therapeutic potential, we characterized the role of PPARγ in sepsis. We analyzed severity of clinical signs, survival rates, cytokine production, leukocyte influx, and bacterial clearance in a cecal ligation and puncture (CLP) model of sepsis in Swiss mice. The PPARγ agonist rosiglitazone treatment improved clinical status and mortality, while increasing IL-10 production and decreasing TNF-α and IL-6 levels, and peritoneal neutrophil accumulation 24 h after CLP. We noted increased bacterial killing in rosiglitazone treated mice, correlated with increased generation of reactive oxygen species. Polymorphonuclear leukocytes (PMN) incubated with LPS or Escherichia coli and rosiglitazone increased peritoneal neutrophil extracellular trap (NET)-mediated bacterial killing, an effect reversed by the PPARγ antagonist (GW 9662) treatment. Rosiglitazone also enhanced the release of histones by PMN, a surrogate marker of NET formation, effect abolished by GW 9662. Rosiglitazone modulated the inflammatory response and increased bacterial clearance through PPARγ activation and NET formation, combining immunomodulatory and host-dependent anti-bacterial effects and, therefore, warrants further study as a potential therapeutic agent in sepsis. PMID:26618986

  12. Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix.

    PubMed

    Yang, Guang; Rothrauff, Benjamin B; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G; Tuan, Rocky S

    2013-12-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM-supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

  13. Phosphorus removal in an enhanced biological phosphorus removal process: roles of extracellular polymeric substances.

    PubMed

    Zhang, Hai-Ling; Fang, Wei; Wang, Yong-Peng; Sheng, Guo-Ping; Zeng, Raymond J; Li, Wen-Wei; Yu, Han-Qing

    2013-10-15

    Phosphorus-accumulating organisms are considered to be the key microorganisms in the enhanced biological phosphorus removal (EBPR) process. A large amount of phosphorus is found in the extracellular polymeric substances (EPS) matrix of these microorganisms. However, the roles of EPS in phosphorus removal have not been fully understood. In this study, the phosphorus in the EBPR sludge was fractionated and further analyzed using quantitative (31)P nuclear magnetic resonance spectroscopy. The amounts and forms of phosphorus in EPS as well as their changes in an anaerobic-aerobic process were also investigated. EPS could act as a reservoir for phosphorus in the anaerobic-aerobic process. About 5-9% of phosphorus in sludge was reserved in the EPS at the end of the aerobic phase and might further contribute to the phosphorus removal. The chain length of the intracellular long-chain polyphosphate (polyP) decreased in the anaerobic phase and then recovered under aerobic conditions. However, the polyP in the EPS had a much shorter chain length than the intracellular polyP in the whole cycle. The migration and transformation of various forms of phosphorus among microbial cells, EPS, and bulk liquid were also explored. On the basis of these results, a model with a consideration of the roles of EPS was proposed, which is beneficial to elucidate the mechanism of phosphorus removal in the EBPR system.

  14. Understanding the role of extracellular polymeric substances in an enhanced biological phosphorus removal granular sludge system.

    PubMed

    Wang, Randeng; Peng, Yongzhen; Cheng, Zhanli; Ren, Nanqi

    2014-10-01

    The role of extracellular polymeric substances (EPS) in the enhanced biological phosphorus removal (EBPR) process was investigated in a P-accumulating granular sludge system by analyzing the distribution and transfer of P, K(+), Mg(2+) and Ca(2+) in the sludge phase, EPS, and the bulk liquid. In the sludge phase, about 30% P, 44.7% K(+), 27.7% Mg(2+), 28% Ca(2+) accumulated in the EPS at the end of aeration. The rate of P, K(+), Mg(2+) and Ca(2+) released from the EPS matrix into the bulk liquid in the anaerobic phase was faster than the rate they were adsorbed from the bulk liquid into the EPS in the aerobic phase. P, K(+), Mg(2+) and Ca(2+) were retained in EPS before transferring into the phosphorus accumulating organisms (PAOs). These results suggest that EPS play a critical role in facilitating the accumulation and transfer of P, K(+), Ca(2+) and Mg(2+) between PAO cells and bulk liquid.

  15. Understanding the role of extracellular polymeric substances in an enhanced biological phosphorus removal granular sludge system.

    PubMed

    Wang, Randeng; Peng, Yongzhen; Cheng, Zhanli; Ren, Nanqi

    2014-10-01

    The role of extracellular polymeric substances (EPS) in the enhanced biological phosphorus removal (EBPR) process was investigated in a P-accumulating granular sludge system by analyzing the distribution and transfer of P, K(+), Mg(2+) and Ca(2+) in the sludge phase, EPS, and the bulk liquid. In the sludge phase, about 30% P, 44.7% K(+), 27.7% Mg(2+), 28% Ca(2+) accumulated in the EPS at the end of aeration. The rate of P, K(+), Mg(2+) and Ca(2+) released from the EPS matrix into the bulk liquid in the anaerobic phase was faster than the rate they were adsorbed from the bulk liquid into the EPS in the aerobic phase. P, K(+), Mg(2+) and Ca(2+) were retained in EPS before transferring into the phosphorus accumulating organisms (PAOs). These results suggest that EPS play a critical role in facilitating the accumulation and transfer of P, K(+), Ca(2+) and Mg(2+) between PAO cells and bulk liquid. PMID:25063972

  16. Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus.

    PubMed

    Banik, Samudra Prosad; Mukherjee, Soumya; Pal, Swagata; Ghorai, Shakuntala; Majumder, Rajib; Khowala, Suman

    2015-01-01

    Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and β-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-β-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.

  17. Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.

    PubMed

    Chen, Hailong; Wang, Zhou; Wang, Zhilai; Dou, Jie; Zhou, Changlin

    2016-04-01

    S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.

  18. Performance of Rodent Spermatozoa Over Time Is Enhanced by Increased ATP Concentrations: The Role of Sperm Competition.

    PubMed

    Tourmente, Maximiliano; Villar-Moya, Pilar; Varea-Sánchez, María; Luque-Larena, Juan J; Rial, Eduardo; Roldan, Eduardo R S

    2015-09-01

    Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa. PMID:26157072

  19. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

    PubMed Central

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-01-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  20. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    PubMed

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  1. 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.

  2. Endochondral Ossification for Enhancing Bone Regeneration: Converging Native Extracellular Matrix Biomaterials and Developmental Engineering In Vivo

    PubMed Central

    Dennis, S. Connor; Berkland, Cory J.; Bonewald, Lynda F.

    2015-01-01

    Autologous bone grafting (ABG) remains entrenched as the gold standard of treatment in bone regenerative surgery. Consequently, many marginally successful bone tissue engineering strategies have focused on mimicking portions of ABG's “ideal” osteoconductive, osteoinductive, and osteogenic composition resembling the late reparative stage extracellular matrix (ECM) in bone fracture repair, also known as the “hard” or “bony” callus. An alternative, less common approach that has emerged in the last decade harnesses endochondral (EC) ossification through developmental engineering principles, which acknowledges that the molecular and cellular mechanisms involved in developmental skeletogenesis, specifically EC ossification, are closely paralleled during native bone healing. EC ossification naturally occurs during the majority of bone fractures and, thus, can potentially be utilized to enhance bone regeneration for nearly any orthopedic indication, especially in avascular critical-sized defects where hypoxic conditions favor initial chondrogenesis instead of direct intramembranous ossification. The body's native EC ossification response, however, is not capable of regenerating critical-sized defects without intervention. We propose that an underexplored potential exists to regenerate bone through the native EC ossification response by utilizing strategies which mimic the initial inflammatory or fibrocartilaginous ECM (i.e., “pro-” or “soft” callus) observed in the early reparative stage of bone fracture repair. To date, the majority of strategies utilizing this approach rely on clinically burdensome in vitro cell expansion protocols. This review will focus on the confluence of two evolving areas, (1) native ECM biomaterials and (2) developmental engineering, which will attempt to overcome the technical, business, and regulatory challenges that persist in the area of bone regeneration. Significant attention will be given to native “raw” materials

  3. Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia

    PubMed Central

    Koyanagi, Satoru; Kusunose, Naoki; Taniguchi, Marie; Akamine, Takahiro; Kanado, Yuki; Ozono, Yui; Masuda, Takahiro; Kohro, Yuta; Matsunaga, Naoya; Tsuda, Makoto; Salter, Michael W.; Inoue, Kazuhide; Ohdo, Shigehiro

    2016-01-01

    Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain. PMID:27739425

  4. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

    Peters, Theodore W; Miller, Aaron W; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E

    2015-11-19

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes.

  5. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

    SciTech Connect

    Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M.; Mroue, Rana; Spencer, Virginia A.; Brownfield, Doug; Radisky, Derek C.; Bustamante, Carlos; Bissell, Mina J.

    2008-10-20

    In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

  6. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

    PubMed Central

    Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M; Mroue, Rana; Spencer, Virginia A; Brownfield, Doug; Radisky, Derek C; Bustamante, Carlos; Bissell, Mina J

    2008-01-01

    In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis. PMID:18843297

  7. Extracellular control of intracellular drug release for enhanced safety of anti-cancer chemotherapy

    PubMed Central

    Zhu, Qian; Qi, Haixia; Long, Ziyan; Liu, Shang; Huang, Zhen; Zhang, Junfeng; Wang, Chunming; Dong, Lei

    2016-01-01

    The difficulty of controlling drug release at an intracellular level remains a key challenge for maximising drug safety and efficacy. We demonstrate herein a new, efficient and convenient approach to extracellularly control the intracellular release of doxorubicin (DOX), by designing a delivery system that harnesses the interactions between the system and a particular set of cellular machinery. By simply adding a small-molecule chemical into the cell medium, we could lower the release rate of DOX in the cytosol, and thereby increase its accumulation in the nuclei while decreasing its presence at mitochondria. Delivery of DOX with this system effectively prevented DOX-induced mitochondria damage that is the main mechanism of its toxicity, while exerting the maximum efficacy of this anti-cancer chemotherapeutic agent. The present study sheds light on the design of drug delivery systems for extracellular control of intracellular drug delivery, with immediate therapeutic implications. PMID:27334142

  8. Extracellular control of intracellular drug release for enhanced safety of anti-cancer chemotherapy

    NASA Astrophysics Data System (ADS)

    Zhu, Qian; Qi, Haixia; Long, Ziyan; Liu, Shang; Huang, Zhen; Zhang, Junfeng; Wang, Chunming; Dong, Lei

    2016-06-01

    The difficulty of controlling drug release at an intracellular level remains a key challenge for maximising drug safety and efficacy. We demonstrate herein a new, efficient and convenient approach to extracellularly control the intracellular release of doxorubicin (DOX), by designing a delivery system that harnesses the interactions between the system and a particular set of cellular machinery. By simply adding a small-molecule chemical into the cell medium, we could lower the release rate of DOX in the cytosol, and thereby increase its accumulation in the nuclei while decreasing its presence at mitochondria. Delivery of DOX with this system effectively prevented DOX-induced mitochondria damage that is the main mechanism of its toxicity, while exerting the maximum efficacy of this anti-cancer chemotherapeutic agent. The present study sheds light on the design of drug delivery systems for extracellular control of intracellular drug delivery, with immediate therapeutic implications.

  9. Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC

    PubMed Central

    Hrabětová, Sabina; Masri, Daniel; Tao, Lian; Xiao, Fanrong; Nicholson, Charles

    2009-01-01

    The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient (D*) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D* for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D* for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties. PMID:19546165

  10. ATP ground- and transition-states of bacterial enhancer binding AAA+ ATPases support complex formation with their target protein, σ54.

    PubMed Central

    Chen, Baoyu; Doucleff, Michaeleen; Wemmer, David E.; De Carlo, Sacha; Huang, Hector H.; Nogales, Eva; Hoover, Timothy R.; Kondrashkina, Elena; Guo, Liang; Nixon, B. Tracy

    2009-01-01

    Summary Transcription initiation by the σ54-form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeFx (ground-state mimics), ADP-AlFx (a transition-state mimic) or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeFx state, and from the ADP-AlFx state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to σ54, with ADP-AlFx having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and σ54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase / promoter complex. PMID:17437715

  11. Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

    PubMed

    Dal-Secco, Daniela; Cunha, Thiago M; Freitas, Andressa; Alves-Filho, José Carlos; Souto, Fabrício O; Fukada, Sandra Y; Grespan, Renata; Alencar, Nylane M N; Neto, Alberto F; Rossi, Marcos A; Ferreira, Sérgio H; Hothersall, John S; Cunha, Fernando Q

    2008-09-15

    In this study, we have addressed the role of H(2)S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H(2)S synthesis inhibitors, dl-propargylglycine (PAG) or beta-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H(2)S donors, NaHS or Lawesson's reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-alpha, keratinocyte-derived chemokine, and LTB(4). Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (K(ATP)(+)) channel blocker, glybenclamide. Conversely, diazoxide, a K(ATP)(+) channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H(2)S augments neutrophil adhesion and locomotion, by a mechanism dependent on K(ATP)(+) channels. PMID:18768887

  12. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    PubMed

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  13. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    PubMed Central

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  14. Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects.

    PubMed

    Altincicek, Boran; Stötzel, Sabine; Wygrecka, Malgorzata; Preissner, Klaus T; Vilcinskas, Andreas

    2008-08-15

    Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses. PMID:18684961

  15. Extracellular miR-1246 promotes lung cancer cell proliferation and enhances radioresistance by directly targeting DR5.

    PubMed

    Yuan, Dexiao; Xu, Jinping; Wang, Juan; Pan, Yan; Fu, Jiamei; Bai, Yang; Zhang, Jianghong; Shao, Chunlin

    2016-05-31

    MiRNAs in the circulation have been demonstrated to be a type of signaling molecule involved in intercellular communication but little is known about their role in regulating radiosensitivity. This study aims to investigate the effects of extracellular miRNAs induced by ionizing radiation (IR) on cell proliferation and radiosensitivity. The miRNAs in the conditioned medium (CM) from irradiated and non-irradiated A549 lung cancer cells were compared using a microarray assay and the profiles of 21 miRNAs up and down-regulated by radiation were confirmed by qRT-PCR. One of these miRNAs, miR-1246, was especially abundant outside the cells and had a much higher level compared with that inside of cells. The expressions of miR-1246 in both A549 and H446 cells increased along with irradiation dose and the time post-irradiation. By labeling exosomes and miR-1246 with different fluorescence dyes, it was found that the extracellular miR-1246 could shuttle from its donor cells to other recipient cells by a non-exosome associated pathway. Moreover, the treatments of cells with miR-1246 mimic or its antisense inhibitor showed that the extracellular miR-1246 could enhance the proliferation and radioresistance of lung cancer cells. A luciferase reporter-gene transfer experiment demonstrated that the death receptor 5 (DR5) was the direct target of miR-1246, and the kinetics of DR5 expression was opposite to that of miR-1246 in the irradiated cells. Our results show that the oncogene-like extracellular miR-1246 could act as a signaling messenger between irradiated and non-irradiated cells, more importantly, it contributes to cell radioresistance by directly suppressing the DR5 gene. PMID:27129166

  16. ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2.

    PubMed

    Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

    2016-02-26

    Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation.

  17. Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production

    PubMed Central

    2012-01-01

    Background A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved. Results Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail. Conclusions The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear

  18. Phenazine production enhances extracellular DNA release via hydrogen peroxide generation in Pseudomonas aeruginosa

    PubMed Central

    Das, Theerthankar; Manefield, Mike

    2013-01-01

    In Pseudomonas aeruginosa eDNA is a crucial component essential for biofilm formation and stability. In this study we report that release of eDNA is influenced by the production of phenazine in P. aeruginosa. A ∆phzA-G mutant of P. aeruginosa PA14 deficient in phenazine production generated significantly less eDNA in comparison with the phenazine producing strains. The relationship between eDNA release and phenazine production is bridged via hydrogen peroxide (H2O2) generation and subsequent H2O2 mediated cell lysis and ultimately release of chromosomal DNA into the extracellular environment as eDNA. PMID:23710274

  19. Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

    PubMed Central

    Laupheimer, Michael; Skorska, Anna; Große, Jana; Tiedemann, Gudrun; Steinhoff, Gustav; David, Robert; Lux, Cornelia A.

    2014-01-01

    Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes. PMID:25610653

  20. Control of a Salmonella virulence locus by an ATP-sensing leader messenger RNA.

    PubMed

    Lee, Eun-Jin; Groisman, Eduardo A

    2012-06-13

    The facultative intracellular pathogen Salmonella enterica resides within a membrane-bound compartment inside macrophages. This compartment must be acidified for Salmonella to survive within macrophages, possibly because acidic pH promotes expression of Salmonella virulence proteins. We reasoned that Salmonella might sense its surroundings have turned acidic not only upon protonation of the extracytoplasmic domain of a protein sensor but also by an increase in cytosolic ATP levels, because conditions that enhance the proton gradient across the bacterial inner membrane stimulate ATP synthesis. Here we report that an increase in cytosolic ATP promotes transcription of the coding region for the virulence gene mgtC, which is the most highly induced horizontally acquired gene when Salmonella is inside macrophages. This transcript is induced both upon media acidification and by physiological conditions that increase ATP levels independently of acidification. ATP is sensed by the coupling/uncoupling of transcription of the unusually long mgtC leader messenger RNA and translation of a short open reading frame located in this region. A mutation in the mgtC leader messenger RNA that eliminates the response to ATP hinders mgtC expression inside macrophages and attenuates Salmonella virulence in mice. Our results define a singular example of an ATP-sensing leader messenger RNA. Moreover, they indicate that pathogens can interpret extracellular cues by the impact they have on cellular metabolites.

  1. A Novel Compound Rasatiol Isolated from Raphanus sativus Has a Potential to Enhance Extracellular Matrix Synthesis in Dermal Fibroblasts

    PubMed Central

    Roh, Seok-Seon; Park, Seung-Bae; Park, Seong-Mo; Choi, Byoung Wook; Lee, Min-Ho; Hwang, Yul-Lye; Kim, Chang Hun; Jeong, Hyun-Ah; Kim, Chang Deok

    2013-01-01

    Background The fibrous proteins of extracellular matrix (ECM) produced by dermal fibroblast contributes to the maintenance of connective tissue integrity. Objective This study is carried out to identify the bioactive ingredient from natural products that enhances ECM production in dermal fibroblasts. Methods Bioassay-directed fractionation was used to isolate the active ingredient from natural extracts. The effects of rasatiol (isolated from Raphanus sativus) on ECM production in primary cultured human dermal fibroblasts was investigated by enzyme linked immunosorbent assay and western blot analysis. Results Rasatiol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen, fibronectin and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was remarkably increased by rasatiol, indicating that enhanced ECM production is linked to the activation of intracellular signaling cascades. Conclusion These results indicate that rasatiol stimulates the fibrous components of ECM production, and may be applied to the maintenance of skin texture. PMID:24003274

  2. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb's cycle flux independent of ATP synthesis

    SciTech Connect

    Cline, Gary W.; Pongratz, Rebecca L.; Zhao, Xiaojian; Papas, Klearchos K.

    2011-11-11

    to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media.

  3. Extracellular secretion of β-glucosidase in ethanologenic E. coli enhances ethanol fermentation of cellobiose.

    PubMed

    Luo, Zichen; Zhang, Yao; Bao, Jie

    2014-09-01

    Consolidated bioprocessing of lignocellulose for ethanol production is realized by expressing cellulase enzymes on ethanologenic strain. In this study, an ethanologenic Escherichia coli ZY81 was constructed by integrating pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis into the genome of E. coli JM109 to obtain the capability of ethanol production. Then, the β-glucosidase gene bglB from Bacillus polymyxa was cloned and secretively expressed in E. coli ZY81. The recombinant strain E. coli ZY81/bglB showed an obvious activity of β-glucosidase in extracellular location with more than half in periplasmic space. EDTA was found to promote the release of the periplasmic proteins by approximately tenfold. E. coli ZY81/bglB utilized cellobiose as sole carbon source for ethanol production with 33.99 % of theoretical yield.

  4. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    PubMed

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  5. ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

    PubMed

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  6. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  7. Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

    PubMed Central

    Samways, Damien S.K.; Egan, Terrance M.

    2007-01-01

    P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors. PMID:17325195

  8. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype.

    PubMed

    Akk, Antonina; Springer, Luke E; Pham, Christine T N

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  9. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade.

  10. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  11. Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

    PubMed

    Iaffaldano, N; Paventi, G; Pizzuto, R; Di Iorio, M; Bailey, J L; Manchisi, A; Passarella, S

    2016-08-01

    This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions. PMID:27036659

  12. Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality.

    PubMed

    Iaffaldano, N; Paventi, G; Pizzuto, R; Di Iorio, M; Bailey, J L; Manchisi, A; Passarella, S

    2016-08-01

    This study examines whether and how helium-neon laser irradiation (at fluences of 3.96-9 J/cm(2)) of cryopreserved ram sperm helps improve semen quality. Pools (n = 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm(2), mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm(2)-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm(2) (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions.

  13. Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator.

    PubMed

    Crane, John K; Olson, Ruth A; Jones, Heather M; Duffey, Michael E

    2002-07-01

    Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea. PMID:12065294

  14. 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. PMID:27295623

  15. 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.

  16. Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections.

    PubMed

    Baelo, Aida; Levato, Riccardo; Julián, Esther; Crespo, Anna; Astola, José; Gavaldà, Joan; Engel, Elisabeth; Mateos-Timoneda, Miguel Angel; Torrents, Eduard

    2015-07-10

    Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections. PMID:25913364

  17. Application of UVA-riboflavin crosslinking to enhance the mechanical properties of extracellular matrix derived hydrogels.

    PubMed

    Ahearne, Mark; Coyle, Aron

    2016-02-01

    Hydrogels derived from extracellular matrix (ECM) have become increasing popular in recent years, particularly for use in tissue engineering. One limitation with ECM hydrogels is that they tend to have poor mechanical properties compared to native tissues they are trying to replicate. To address this problem, a UVA (ultraviolet-A) riboflavin crosslinking technique was applied to ECM hydrogels to determine if it could be used to improve their elastic modulus. Hydrogels fabricated from corneal, cardiac and liver ECM were used in this study. The mechanical properties of the hydrogels were characterized using a spherical indentation technique. The microstructure of the hydrogels and the cytotoxic effect of crosslinking on cell seeded hydrogels were also evaluated. The combination of UVA light and riboflavin solution led to a significant increase in elastic modulus from 6.8kPa to 24.7kPa, 1.4kPa to 6.9kPa and 0.9kPa to 1.6kPa for corneal, cardiac and liver ECM hydrogels respectively. The extent of this increase was dependent on a number of factors including the UVA exposure time and the initial hydrogel concentration. There were also a high percentage of viable cells within the cell seeded hydrogels with 94% of cells remaining viable after 90min exposure to UVA light. These results suggest that UVA-riboflavin crosslinking is an effective approach for improving the mechanical properties of ECM hydrogels without resulting in a significant reduction of cell viability.

  18. Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation.

    PubMed

    Rashid, Rafi; Lim, Natalie Sheng Jie; Chee, Stella Min Ling; Png, Si Ning; Wohland, Thorsten; Raghunath, Michael

    2014-12-01

    Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. Since standard cell culture media lack this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix (ECM) deposition, is impaired in vitro. To bring back physiological crowdedness to in vitro systems, we have previously introduced carbohydrate-based macromolecules to culture media and have achieved marked improvements with mixed MMC in terms of ECM deposition and differentiation of mesenchymal stem cells (MSCs). We show here that although this system is successful, it is limited, due to viscosity, to only 33% of the fractional volume occupancy (FVO) of full serum, which we calculated to have an FVO of approximately 54% v/v. We show here that full-serum FVO can be achieved using polyvinylpyrrolidone (PVP) 360 kDa. Under these conditions, ECM deposition in human fibroblasts and MSCs is on par, if not stronger than, with original MMC protocols using carbohydrates, but with a viscosity that is not significantly changed. In addition, we have found that the proliferation rate for bone marrow-derived MSCs and fibroblasts increases slightly in the presence of PVP360, similar to that observed with carbohydrate-based crowders. A palette of MMC compounds is now emerging that enables us to tune the crowdedness of culture media seamlessly from interstitial fluid (9% FVO), in which the majority of tissue cells might be based, to serum environments mimicking intravascular conditions. Despite identical FVO's, individual crowder size effects play a role and different cell types appear to have preferences in terms of FVO and the crowder that this is achieved with. However, in the quest of crowders that we have predicted to have a smoother regulatory approval path, PVP is a highly interesting compound, as it has been widely used in the medical and food industries and shows a novel promising use in cell culture and

  19. Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections.

    PubMed

    Baelo, Aida; Levato, Riccardo; Julián, Esther; Crespo, Anna; Astola, José; Gavaldà, Joan; Engel, Elisabeth; Mateos-Timoneda, Miguel Angel; Torrents, Eduard

    2015-07-10

    Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections.

  20. Redox regulation of ATP sulfurylase in microalgae.

    PubMed

    Prioretti, Laura; Lebrun, Régine; Gontero, Brigitte; Giordano, Mario

    2016-09-30

    ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS. PMID:27613093

  1. Redox regulation of ATP sulfurylase in microalgae.

    PubMed

    Prioretti, Laura; Lebrun, Régine; Gontero, Brigitte; Giordano, Mario

    2016-09-30

    ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS.

  2. Imaging and characterization of stretch-induced ATP release from alveolar A549 cells

    PubMed Central

    Grygorczyk, Ryszard; Furuya, Kishio; Sokabe, Masahiro

    2013-01-01

    Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin–luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10–40%)-induced transient ATP release from a small fraction (≤1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (≤150 μm) of releasing cells often exceeded 10 μm, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca2+ responses, rapid sustained Ca2+ elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca2+ waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing. PMID:23247110

  3. Imaging and characterization of stretch-induced ATP release from alveolar A549 cells.

    PubMed

    Grygorczyk, Ryszard; Furuya, Kishio; Sokabe, Masahiro

    2013-03-01

    Abstract  Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin-luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10-40%)-induced transient ATP release from a small fraction (1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (150 μm) of releasing cells often exceeded 10 μm, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca(2+) responses, rapid sustained Ca(2+) elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca(2+) waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing.

  4. cAMP/Protein Kinase A Activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP Release from Rat Skeletal Muscle during Low pH or Contractions

    PubMed Central

    Cai, Weisong; Ballard, Heather J.

    2012-01-01

    We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTRinh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTRinh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na+/H+-exchanger (NHE) with amiloride or the Na+/Ca2+-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway. PMID:23226244

  5. Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

    PubMed

    Li, Shuo; Chen, Xiaoli; Hao, Gaixiang; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-03-01

    Nucleotide oligomerization domain (NOD)-like receptor (NLR) family with a caspase activation and recruitment domain (CARD) containing 3 (NLRC3) protein is an important cytosolic pattern recognition receptor that negatively regulates innate immune response in mammals. Hitherto, the immunological significance of NLRC3 protein in fish remains largely uncharacterized. Here we identified and characterized a novel NLRC3 gene (named poNLRC3) implicated in regulation of fish innate immunity from Japanese flounder Paralichthys olivaceus. The poNLRC3 protein is a cytoplasmic protein with an undefined N-terminal domain, a NACHT domain, a fish-specific NACHT associated domain, six LRR motifs, and a C-terminal fish-specific PYR/SPYR (B30.2) domain but only shares less than 40% sequence identities with the known Japanese flounder NLRC proteins. poNLRC3 gene is ubiquitously expressed in all tested tissues and is dominantly expressed in the Japanese flounder head kidney macrophages (HKMs). We for the first time showed that poNLRC3 expression was significantly modulated by the stimulation of extracellular ATP, an important danger/damage-associated molecular pattern in activating innate immunity in P. olivaceus. Importantly, we revealed that poNLRC3 plays an important role in positively regulating ATP-induced IL-1beta and IL-6 gene expression, suggesting the involvement of poNLRC3 in extracellular ATP-mediated immune signaling. In addition, we showed that poNLRC3 mRNA expression was up-regulated in response to LPS and Edwardsiella tarda immune challenges. Finally, we showed that down-regulating the endogenous poNLRC3 expression with small interfering RNA significantly reduced LPS-induced proinflammatory cytokine gene expression in the Japanese flounder HKM cells. Altogether, we have identified a novel inducible fish NLR member, poNLRC3, which is involved in extracellular ATP-mediated immune signaling and may positively regulate the LPS-induced innate immune response in the Japanese

  6. Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death

    PubMed Central

    Draganov, Dobrin; Gopalakrishna-Pillai, Sailesh; Chen, Yun-Ru; Zuckerman, Neta; Moeller, Sara; Wang, Carrie; Ann, David; Lee, Peter P.

    2015-01-01

    Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca2+/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy. PMID:26552848

  7. Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death.

    PubMed

    Draganov, Dobrin; Gopalakrishna-Pillai, Sailesh; Chen, Yun-Ru; Zuckerman, Neta; Moeller, Sara; Wang, Carrie; Ann, David; Lee, Peter P

    2015-01-01

    Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy. PMID:26552848

  8. Neutrophil Extracellular Trap-Associated Protein Activation of the NLRP3 Inflammasome Is Enhanced in Lupus Macrophages

    PubMed Central

    Kahlenberg, J. Michelle; Carmona-Rivera, Carmelo; Smith, Carolyne K.; Kaplan, Mariana J.

    2012-01-01

    Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus (SLE), while NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells and may participate in organ damage through incompletely characterized pathways. In order to better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on the NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2×7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines which further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage. PMID:23267025

  9. Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages.

    PubMed

    Kahlenberg, J Michelle; Carmona-Rivera, Carmelo; Smith, Carolyne K; Kaplan, Mariana J

    2013-02-01

    Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus, and NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells, and may participate in organ damage through incompletely characterized pathways. To better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2X7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines that further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.

  10. Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels.

    PubMed

    Tagler, David; Makanji, Yogeshwar; Tu, Tao; Bernabé, Beatriz Peñalver; Lee, Raymond; Zhu, Jie; Kniazeva, Ekaterina; Hornick, Jessica E; Woodruff, Teresa K; Shea, Lonnie D

    2014-07-01

    The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (<70 µm) rapidly lost structure and degenerated. These morphologic changes were associated with a breakdown of the follicular basement membrane; hence, this study investigated ascorbic acid based on its role in extracellular matrix (ECM) deposition/remodeling for other applications. The selection of ascorbic acid was further supported by a microarray analysis that suggested a decrease in mRNA levels of enzymes within the ascorbate pathway between primordial, primary, and secondary follicles. The supplementation of ascorbic acid (50 µg/mL) significantly enhanced the survival of primary follicles (<80 µm) cultured in alginate hydrogels, which coincided with improved structural integrity. Follicles developed antral cavities and increased to diameters exceeding 250 µm. Consistent with improved structural integrity, the gene/protein expression of ECM and cell adhesion molecules was significantly changed. This research supports the notion that modifying the culture environment (medium components) can substantially enhance the survival and growth of early stage follicles.

  11. Neomycin enhances extracellular matrix stability of glutaraldehyde crosslinked bioprosthetic heart valves.

    PubMed

    Friebe, Vincent M; Mikulis, Brandon; Kole, Sourav; Ruffing, Christy S; Sacks, Michael S; Vyavahare, Naren R

    2011-11-01

    Glutaraldehyde (GLUT) crosslinked porcine aortic heart valves are continued to be extensively used in heart valve replacement surgeries. GLUT does not crosslink glycosaminoglycans in the tissue and we have demonstrated that GAG loss is associated with tissue degeneration. In this study, we examined the ability of neomycin to enhance GLUT crosslinking to stabilize GAGs, as well as provide evidence of improved functional integrity. Neomycin enhanced GLUT crosslinked (NG) leaflets exposed to collagenase and elastase enzymes exhibited an increased resistance to proteolytic degradation. Furthermore, NG leaflets exhibited small but significant increases in collagen denaturation temperatures when compared to that of standard GLUT crosslinked BHVs. NG leaflets subjected to storage, accelerated cyclic fatigue, and in vitro enzyme mediated GAG degradation revealed improved GAG stabilization versus standard GLUT crosslinked valves, which sustained substantial decreases in GAG content. Ultrastructural analysis using transmission electron microscopy qualitatively confirmed NG leaflets preserved GAGs after enzymatic degradation. Biomechanical analyses demonstrated that NG leaflets were functionally similar to GLUT tissues but were slightly stiffer under both planar biaxial tension and under flexure. Interestingly, after GAGase treatment, GLUT tissues showed increased areal compliance and reduced hysteresis, while NG leaflets were unchanged. Collectively, NG cross-linking functionally insulated the tissue from GAG digestion, and imparted modest additional matrix stiffness but maintained tissue hysteresis properties.

  12. Parathyroid hormone 1-34 enhances extracellular matrix deposition and organization during flexor tendon repair.

    PubMed

    Lee, Daniel J; Southgate, Richard D; Farhat, Youssef M; Loiselle, Alayna E; Hammert, Warren C; Awad, Hani A; O'Keefe, Regis J

    2015-01-01

    Parathyroid hormone (PTH) 1-34 is known to enhance fracture healing. Tendon repair is analogous to bone healing in its dependence on the proliferation and differentiation of mesenchymal stem cells, matrix formation, and tissue remodeling.(1,2,3) We hypothesized that PTH 1-34 enhances tendon healing in a flexor digitorum longus (FDL) tendon repair model. C57Bl/6J mice were treated with either intraperitoneal PTH 1-34 or vehicle-control (PBS). Tendons were harvested at 3-28 days for histology, gene expression, and biomechanical testing. The metatarsophalangeal joint range of motion was reduced 1.5-2-fold in PTH 1-34 mice compared to control mice. The gliding coefficient, a measure of adhesion formation, was 2-3.5-fold higher in PTH 1-34 mice. At 14 days post-repair, the tensile strength was twofold higher in PTH 1-34 specimens, but at 28 days there were no differences. PTH 1-34 mice had increased fibrous tissue deposition that correlated with elevated expression of collagens and fibronectin as seen on quantitative PCR. PTH 1-34 accelerated the deposition of reparative tissue but increased adhesion formation.

  13. An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.

    PubMed

    Bao, Ru-Meng; Yang, Hong-Ming; Yu, Chang-Mei; Zhang, Wei-Fen; Tang, Jin-Bao

    2016-10-01

    Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the β-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.

  14. Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

    PubMed

    Francis, Brian R; White, Karen H; Thorsness, Peter E

    2007-04-01

    ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

  15. miR-495 enhances the sensitivity of non-small cell lung cancer cells to platinum by modulation of copper-transporting P-type adenosine triphosphatase A (ATP7A).

    PubMed

    Song, Liqiang; Li, Yan; Li, Weina; Wu, Shouzhen; Li, Zhikui

    2014-07-01

    Copper-transporting P-type adenosine triphosphatase A (ATP7A) is associated with platinum drug resistance in non-small cell lung cancer (NSCLC). microRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression at post-transcriptional level. In this study, the aim is to explore which miRNAs might participate in the platinum resistance by targeting ATP7A in NSCLC. Using real-time PCR-based miRNA expression profiling and bioinformatics, we selected miR-495 as a candidate miRNA. EGFP reporter assay, real-time PCR, and Western blot validated that ATP7A was a direct target for miR-495. The drug sensitivity assay indicated that miR-495 enhanced the cell response to cisplatin (CDDP) in NSCLC cells, while inhibition of miR-495 led to the opposite effects. Importantly, either overexpression or knockdown of ATP7A could override the effect of miR-495 on chemosensitivity. We also demonstrated that miR-495 increased the intracellular CDDP accumulation and overexpression of ATP7A can reduce the increased drug concentration induced by miR-495. Finally, we discovered that there was a converse relationship between miR-495 and ATP7A levels in NSCLC tissues sensitive or resistant to CDDP. In conclusion, our data demonstrate that miR-495 regulates the multi-drug resistance by modulation of ATP7A expression in NSCLC and suggest that miR-495 may serve as a potential biomarker for the treatment of multi-drug resistant NSCLC patients with high ATP7A levels. PMID:24038379

  16. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels.

    PubMed

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-10-15

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mM) with a significant increase in [ATP](c). Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mM glucose with increases in [ATP](c). Further implicating an ATP-independent mechanism of K(ATP) channel closure in hypothalamic neurons, removal of extracellular glucose (10 mM) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mM) of intracellular ATP. Neurons from both brain regions responded to 5 mM lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP](c). High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed K(ATP) channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.

  17. Metabolic Agents that Enhance ATP can Improve Cognitive Functioning: A Review of the Evidence for Glucose, Oxygen, Pyruvate, Creatine, and L-Carnitine

    PubMed Central

    Owen, Lauren; Sunram-Lea, Sandra I.

    2011-01-01

    Over the past four or five decades, there has been increasing interest in the neurochemical regulation of cognition. This field received considerable attention in the 1980s, with the identification of possible cognition enhancing agents or “smart drugs”. Even though many of the optimistic claims for some agents have proven premature, evidence suggests that several metabolic agents may prove to be effective in improving and preserving cognitive performance and may lead to better cognitive aging through the lifespan. Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. There are a number of agents with the potential to improve metabolic activity. Research is now beginning to identify these various agents and delineate their potential usefulness for improving cognition in health and disease. This review provides a brief overview of the metabolic agents glucose, oxygen, pyruvate, creatine, and L-carnitine and their beneficial effects on cognitive function. These agents are directly responsible for generating ATP (adenosine triphosphate) the main cellular currency of energy. The brain is the most metabolically active organ in the body and as such is particularly vulnerable to disruption of energy resources. Therefore interventions that sustain adenosine triphosphate (ATP) levels may have importance for improving neuronal dysfunction and loss. Moreover, recently, it has been observed that environmental conditions and diet can affect transgenerational gene expression via epigenetic mechanisms. Metabolic agents might play a role in regulation of nutritional epigenetic effects. In summary, the reviewed metabolic agents represent a promising strategy for improving cognitive function and possibly slowing or preventing cognitive decline. PMID:22254121

  18. Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.

    PubMed

    Liu, Dan; Zhang, Zheng; Liman, Emily R

    2005-05-27

    TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

  19. Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    PubMed Central

    Ludtmann, Marthe H.R.; Angelova, Plamena R.; Ninkina, Natalia N.; Gandhi, Sonia

    2016-01-01

    Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance

  20. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    PubMed Central

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C. J.; Raaijmakers, Marc H.; van Wijnen, Andre J.; Cornelissen, Jan J.; van Leeuwen, Johannes P.

    2016-01-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders. PMID:27585950

  1. 6-demethoxynobiletin, a nobiletin-analog citrus flavonoid, enhances extracellular signal-regulated kinase phosphorylation in PC12D cells.

    PubMed

    Kimura, Junko; Nemoto, Kiyomitsu; Yokosuka, Akihito; Mimaki, Yoshihiro; Degawa, Masakuni; Ohizumi, Yasushi

    2013-01-01

    We previously demonstrated that nobiletin, a polymethoxylated flavone isolated from citrus peels, has the potential to improve cognitive dysfunction in patients with Alzheimer's disease (AD). Recent studies suggest that the generation of intraneuronal amyloid-beta (Aβ) oligomers is an early event in the pathogenesis of AD. Aβ oligomers cause deficits in the regulation of the extracellular signal-regulated kinase (ERK) signaling which is critical for consolidation of the memory. Our previous studies revealed that nobiletin activated ERK signaling and subsequent cyclic AMP response element-dependent transcription. In this study, the effects of five nobiletin analogs, 6-demethoxynobiletin, tangeretin, 5-demethylnobiletin, sinensetin, and 6-demethoxytangeretin, isolated from citrus peels were assessed on ERK phosphorylation in PC12D cells, and the structure-activity relationships were examined. PC12D cells were treated with nobiletin or its analogs, and the cell extracts were analyzed by Western blotting using an antibody specific to phosphorylated ERK. 6-Demethoxynobiletin markedly enhanced ERK phosphorylation in a concentration-dependent manner. These results may be useful in developing drugs and functional foods using citrus peels for the treatment of dementia including AD.

  2. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials. PMID:25637448

  3. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.

  4. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells

    NASA Astrophysics Data System (ADS)

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W. J. C.; Ter Borg, Mariette N. D.; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C. J.; Raaijmakers, Marc H.; van Wijnen, Andre J.; Cornelissen, Jan J.; van Leeuwen, Johannes P.

    2016-09-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.

  5. Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells.

    PubMed

    Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric; Rombouts, Elwin W J C; Ter Borg, Mariette N D; Dudakovic, Amel; Chiba, Hideki; van der Eerden, Bram C J; Raaijmakers, Marc H; van Wijnen, Andre J; Cornelissen, Jan J; van Leeuwen, Johannes P

    2016-01-01

    Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Using next-generation sequencing we show that human osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of human umbilical cord blood-derived CD34(+) HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34(+) cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and successfully engraft in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders. PMID:27585950

  6. Extracellular matrix-regulated neural differentiation of human multipotent marrow progenitor cells enhances functional recovery after spinal cord injury

    PubMed Central

    Deng, Win-Ping; Yang, Chi-Chiang; Yang, Liang-Yo; Chen, Chun-Wei D.; Chen, Wei-Hong; Yang, Charn-Bing; Chen, Yu-Hsin; Lai, Wen-Fu T.; Renshaw, Perry F.

    2015-01-01

    BACKGROUND CONTEXT Recent advanced studies have demonstrated that cytokines and extracellular matrix (ECM) could trigger various types of neural differentiation. However, the efficacy of differentiation and in vivo transplantation has not yet thoroughly been investigated. PURPOSE To highlight the current understanding of the effects of ECM on neural differentiation of human bone marrow-derived multipotent progenitor cells (MPCs), regarding state-of-art cure for the animal with acute spinal cord injury (SCI), and explore future treatments aimed at neural repair. STUDY DESIGN A selective overview of the literature pertaining to the neural differentiation of the MSCs and experimental animals aimed at improved repair of SCI. METHODS Extracellular matrix proteins, tenascin-cytotactin (TN-C), tenascin-restrictin (TN-R), and chondroitin sulfate (CS), with the cytokines, nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF)/retinoic acid (RA) (NBR), were incorporated to induce transdifferentiation of human MPCs. Cells were treated with NBR for 7 days, and then TN-C, TN-R, or CS was added for 2 days. The medium was changed every 2 days. Twenty-four animals were randomly assigned to four groups with six animals in each group: one experimental and three controls. Animals received two (bilateral) injections of vehicle, MPCs, NBR-induced MPCs, or NBR/TN-C-induced MPCs into the lesion sites after SCI. Functional assessment was measured using the Basso, Beattie, and Bresnahan locomotor rating score. Data were analyzed using analysis of variance followed by Student-Newman-Keuls (SNK) post hoc tests. RESULTS Results showed that MPCs with the transdifferentiation of human MPCs to neurons were associated with increased messenger-RNA (mRNA) expression of neuronal markers including nestin, microtubule-associated protein (MAP) 2, glial fibrillary acidic protein, βIII tubulin, and NGF. Greater amounts of neuronal morphology appeared in cultures incorporated with TN-C and TN

  7. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    SciTech Connect

    Zhang, Yun

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  8. Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

    PubMed Central

    Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

    2015-01-01

    Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that

  9. External Dentin Stimulation Induces ATP Release in Human Teeth.

    PubMed

    Liu, X; Wang, C; Fujita, T; Malmstrom, H S; Nedergaard, M; Ren, Y F; Dirksen, R T

    2015-09-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain. PMID:26130258

  10. External Dentin Stimulation Induces ATP Release in Human Teeth

    PubMed Central

    Wang, C.; Fujita, T.; Malmstrom, H.S.; Nedergaard, M.; Ren, Y.F.; Dirksen, R.T.

    2015-01-01

    ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation–induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation–induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain. PMID:26130258

  11. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    PubMed

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients.

  12. Spare mitochondrial respiratory capacity permits human adipocytes to maintain ATP homeostasis under hypoglycemic conditions.

    PubMed

    Keuper, Michaela; Jastroch, Martin; Yi, Chun-Xia; Fischer-Posovszky, Pamela; Wabitsch, Martin; Tschöp, Matthias H; Hofmann, Susanna M

    2014-02-01

    Mitochondrial dysfunction in white adipose tissue plays a key role in the pathogenesis of type 2 diabetes. Emerging evidence specifically suggests that altered oxidative phosphorylation in adipocytes may have a relevant effect on systemic glucose homeostasis, requiring understanding of adipocyte bioenergetics. We analyzed energetic flux of an intact human adipocyte cell model by plate-based respirometry and extracellular acidification. During differentiation, we discovered that glycolytic ATP production was increasingly replaced by mitochondrial oxidative metabolism (from 20 to 60%). This observation was corroborated by simultaneous up-regulation of canonical mitochondrial gene programs, such as peroxisome proliferator-activated receptor γ coactivator α (PGC1α; 150-fold) and cytochrome c-1 (CytC; 3-fold). Mimicking diabetic phenotypes by exposure to various glucose levels (0, 5, and 25 mM) resulted in immediate adjustments of glycolytic and mitochondrial activity that aimed to maintain intracellular ATP. We conclude that ATP deficits by mitochondrial failure are compensated by glycolytic ATP production, resulting in inefficient conversion of glucose to cellular ATP. Metabolic inefficiency may enhance glucose uptake, therefore improving systemic glucose homeostasis. Notably, mature adipocytes developed a high spare respiratory capacity (increased by 6-fold) permitting rapid adaptation to metabolic changes. Spare respiratory capacity may also allow additional metabolic scope for energy dissipation, potentially offering new therapeutic targets for the treatment of metabolic disease.

  13. Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression.

    PubMed

    Mori, Miyuki; Kasa, Shojiro; Isozaki, Yoshihiro; Kamori, Tsugumitsu; Yamaguchi, Shoichiro; Ueda, Shuji; Kuwano, Toshio; Eguchi, Minako; Isayama, Keishiro; Nishimura, Shotaro; Tabata, Shoji; Yamauchi, Nobuhiko; Hattori, Masa-aki

    2013-01-01

    The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression. PMID:23164983

  14. Cellular Pathophysiology of an Adrenal Adenoma-Associated Mutant of the Plasma Membrane Ca(2+)-ATPase ATP2B3.

    PubMed

    Tauber, Philipp; Aichinger, B; Christ, C; Stindl, J; Rhayem, Y; Beuschlein, F; Warth, R; Bandulik, S

    2016-06-01

    Adrenal aldosterone-producing adenomas (APAs) are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. These stimuli lead to a depolarization of the plasma membrane and, as a consequence, an increase of intracellular Ca(2+). Mutations of the plasma membrane Ca(2+)-ATPase ATP2B3 have been found in APAs with a prevalence of 0.6%-3.1%. Here, we investigated the effects of the APA-associated ATP2B3(Leu425_Val426del) mutation in adrenocortical NCI-H295R and human embryonic kidney (HEK-293) cells. Ca(2+) measurements revealed a higher basal Ca(2+) level in cells expressing the mutant ATP2B3. This rise in intracellular Ca(2+) was even more pronounced under conditions with high extracellular Ca(2+) pointing to an increased Ca(2+) influx associated with the mutated protein. Furthermore, cells with the mutant ATP2B3 appeared to have a reduced capacity to export Ca(2+) suggesting a loss of the physiological pump function. Surprisingly, expression of the mutant ATP2B3 caused a Na(+)-dependent inward current that strongly depolarized the plasma membrane and compromised the cytosolic cation composition. In parallel to these findings, mRNA expression of the cytochrome P450, family 11, subfamily B, polypeptide 2 (aldosterone synthase) was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3. In summary, the APA-associated ATP2B3(Leu425_Val426del) mutant promotes aldosterone production by at least 2 different mechanisms: 1) a reduced Ca(2+) export due to the loss of the physiological pump function; and 2) an increased Ca(2+) influx due to opening of depolarization-activated Ca(2+) channels as well as a possible Ca(2+) leak through the mutated pump. PMID:27035656

  15. Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

    PubMed

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V; Schuetz, John D

    2013-09-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  16. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  17. Shockwaves increase T-cell proliferation and IL-2 expression through ATP release, P2X7 receptors, and FAK activation.

    PubMed

    Yu, Tiecheng; Junger, Wolfgang G; Yuan, Changji; Jin, An; Zhao, Yi; Zheng, Xueqing; Zeng, Yanjun; Liu, Jianguo

    2010-03-01

    Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/mm(2) induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation.

  18. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

    PubMed Central

    2011-01-01

    Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by

  19. Paclitaxel-Loaded Mixed Micelles Enhance Ovarian Cancer Therapy through Extracellular pH-Triggered PEG Detachment and Endosomal Escape.

    PubMed

    Zhao, Haijun; Li, Qian; Hong, Zehui

    2016-07-01

    Although PEGylation allows a drug delivery vehicle to have prolonged blood circulation time, it faces the problem of reduced cellular uptake. Removal of the polyethylene glycol (PEG)-shell at the appropriate time through tumor-microenvironment triggers could be a feasible solution to this problem. Here, paclitaxel (PTX)-loaded mixed micelles (PTX-mM) self-assembled from stearate-modified hyaluronic acid (SHA), mPEG-b-poly(β-amino ester) (mPEG-b-PAE), and ethylene acetyl-b-poly(β-amino ester) (EA-b-PAE) were developed. In the preparation of PTX-mM, SHA micelles were coated with EA-b-PAE followed by coloading of PTX and mPEG-b-PAE. PTX-mM were capable of extracellular pH-triggered PEG-detachment and poly(β-amino ester) (PAE)-mediated endosomal escape. When the pH was changed from pH 7.4 to pH 6.8, the particle size of PTX-mM significantly decreased from 97.5 ± 4.4 to 71.5 ± 2.3 nm. It also resulted in rapid and complete release of mPEG-b-PAE from PTX-mM as monitored using quartz crystal microbalance (QCM) technology. PTX-mM capable of PEG detachment provided significant enhancement of PTX accumulation in SKOV-3 cells compared to PEG nondetachable PTX-mM. Interestingly, intracellular transport studies using confocal laser scanning microscopy (CLSM) showed that EA-b-PAE could promote the escape of micelles from endolysosomes. The half-maximal inhibitory concentration (IC50) of PTX-mM against SKOV-3 cells was 5.7 μg/mL, and PTX-mM containing 20 μg/mL of PTX induced apoptosis in 53.0% of the cell population. PTX-mM exhibited a highly prolonged elimination half-life (t1/2, 2.83 ± 0.37 h) and improved area under the curve (AUC, 7724.82 ± 1190.75 ng/mL/h) than the PTX-loaded SHA micelles (PTX-M). Furthermore, PTX-mM showed the highest tumor inhibition rate (64.9%) and the longest survival time (53 days) against the SKOV-3 ovarian cancer xenograft models among all formulations. Taken together, the results suggested that PTX-mM have potential as an efficient

  20. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K+ channels

    PubMed Central

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-01-01

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25–luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP-independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP]c. High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells. PMID:12381816

  1. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

  2. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities.

  3. The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor.

    PubMed

    Yegutkin, Gennady G; Mikhailov, Andrey; Samburski, Sergei S; Jalkanen, Sirpa

    2006-08-01

    Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

  4. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-08-19

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease.

  5. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation

    PubMed Central

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer’s disease. PMID:27548242

  6. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease. PMID:27548242

  7. Extracellular matrix alterations, accelerated leukocyte infiltration and enhanced axonal sprouting after spinal cord hemisection in tenascin-C-deficient mice.

    PubMed

    Schreiber, Jenny; Schachner, Melitta; Schumacher, Udo; Lorke, Dietrich Ernst

    2013-10-01

    The extracellular matrix glycoprotein tenascin-C has been implicated in wound repair and axonal growth. Its role in mammalian spinal cord injury is largely unknown. In vitro it can be both neurite-outgrowth promoting and repellent. To assess its effects on glial reactions, extracellular matrix formation, and axonal regrowth/sprouting in vivo, 20 tenascin-C-deficient and 20 wild type control mice underwent lumbar spinal cord hemisection. One, three, seven and fourteen days post-surgery, cryostat sections of the spinal cord were examined by conventional histology and by immunohistochemistry using antibodies against F4/80 (microglia/macrophage), GFAP (astroglia), neurofilament, fibronectin, laminin and collagen type IV. Fibronectin immunoreactivity was significantly down-regulated in tenascin-C-deficient mice. Moreover, fourteen days after injury, immunodensity of neurofilament-positive fibers was two orders of magnitude higher along the incision edges of tenascin-C-deficient mice as compared to control mice. In addition, lymphocyte infiltration was seen two days earlier in tenascin-C-deficient mice than in control mice and neutrophil infiltration was increased seven days after injury. The increase in thin neurofilament positive fibers in tenascin-C-deficient mice indicates that lack of tenascin-C alters the inflammatory reaction and extracellular matrix composition in a way that penetration of axonal fibers into spinal cord scar tissue may be facilitated.

  8. Enhancement of the dewaterability of sludge during bioleaching mainly controlled by microbial quantity change and the decrease of slime extracellular polymeric substances content.

    PubMed

    Huo, Minbo; Zheng, Guanyu; Zhou, Lixiang

    2014-09-01

    Contribution rates of factors controlling sludge dewaterability during bioleaching, such as sludge pH, microbial quantity, extracellular polymeric substances (EPS), etc., were investigated in this study. Results showed that the dewaterability of bioleached sludge was jointly enhanced by the growth of Acidithiobacillus sp., the increase of Fe(3+) concentration, the decreases of sludge pH, heterotrophic microorganism quantity change, and the decreases of EPS and bound water contents. Ridge regression analysis further revealed that the contribution rates of microbial quantity change, bound water content and slime EPS content on sludge dewaterability enhancement were 32.50%, 24.24%, and 22.37%, respectively, all of which are dominant factors. Therefore, the enhancement of sludge dewaterability was mainly controlled by microbial quantity change and the decrease of bound water and slime EPS contents during bioleaching.

  9. Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

    PubMed Central

    McNicholas, C M; Guggino, W B; Schwiebert, E M; Hebert, S C; Giebisch, G; Egan, M E

    1996-01-01

    We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels. PMID:8755607

  10. The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)▿

    PubMed Central

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-01-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P2 variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  11. The second extracellular loop of pore-forming subunits of ATP-binding cassette transporters for basic amino acids plays a crucial role in interaction with the cognate solute binding protein(s).

    PubMed

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-04-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)(2) complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP(2) of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P(2) variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  12. 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.

  13. Physiological levels of ATP Negatively Regulate Proteasome Function

    PubMed Central

    Huang, Hongbiao; Zhang, Xiaoyan; Li, Shujue; Liu, Ningning; Lian, Wen; McDowell, Emily; Zhou, Ping; Zhao, Canguo; Guo, Haiping; Zhang, Change; Yang, Changshan; Wen, Guangmei; Dong, Xiaoxian; Lu, Li; Ma, Ningfang; Dong, Weihua; Dou, Q. Ping; Wang, Xuejun; Liu, Jinbao

    2010-01-01

    Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50–100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions. PMID:20805844

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

    SciTech Connect

    Jason Alan Gruenhagen

    2003-12-12

    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. Ca{sup 2+} imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca{sup 2+} 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{sup +} 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 functionalized Cd

  15. Disulfiram anti-cancer efficacy without copper overload is enhanced by extracellular H2O2 generation: antagonism by tetrathiomolybdate

    PubMed Central

    Calderon-Aparicio, Ali; Strasberg-Rieber, Mary; Rieber, Manuel

    2015-01-01

    Highlights exogenous SOD increases apoptosis by sub-toxic disulfiram without copper overload H2O2 generation from glucose oxidase also potentiates disulfiram toxicity N-acetylcysteine suppresses antitumor potentiation of DSF by H2O2 generation sub-toxic tetrathiomolybdate inhibits potentiation of DSF by SOD Background Cu/Zn superoxide dismutases (SODs) like the extracellular SOD3 and cytoplasmic SOD1 regulate cell proliferation by generating hydrogen peroxide (H2O2). This pro-oxidant inactivates essential cysteine residues in protein tyrosine phosphatases (PTP) helping receptor tyrosine kinase activation by growth factor signaling, and further promoting downstream MEK/ERK linked cell proliferation. Disulfiram (DSF), currently in clinical cancer trials is activated by copper chelation, being potentially capable of diminishing the copper dependent activation of MEK1/2 and SOD1/SOD3 and promoting reactive oxygen species (ROS) toxicity. However, copper (Cu) overload may occur when co-administered with DSF, resulting in toxicity and mutagenicity against normal tissue, through generation of the hydroxyl radical (•OH) by the Fenton reaction. Purpose To investigate: a) whether sub-toxic DSF efficacy can be increased without Cu overload against human melanoma cells with unequal BRAF(V600E) mutant status and Her2-overexpressing SKBR3 breast cancer cells, by increasing H2O2from exogenous SOD; b) to compare the anti-tumor efficacy of DSF with that of another clinically used copper chelator, tetrathiomolybdate (TTM) Results a) without copper supplementation, exogenous SOD potentiated sub-toxic DSF toxicity antagonized by sub-toxic TTM or by the anti-oxidant N-acetylcysteine; b) exogenous glucose oxidase, another H2O2 generator resembled exogenous SOD in potentiating sub-toxic DSF. Conclusions potentiation of sub-lethal DSF toxicity by extracellular H2O2 against the human tumor cell lines investigated, only requires basal Cu and increased ROS production, being unrelated to non

  16. 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

  17. Generation of priming mesenchymal stem cells with enhanced potential to differentiate into specific cell lineages using extracellular matrix proteins.

    PubMed

    Han, Na Rae; Yun, Jung Im; Park, Young Hyun; Ahn, Ji Yeon; Kim, Choonghyo; Choi, Jung Hoon; Lee, Eunsong; Lim, Jeong Mook; Lee, Seung Tae

    2013-07-01

    Poor understanding of the differentiation of mesenchymal stem cells (MSCs) has resulted in a low differentiation yield, and has hindered their application in medicine. As a solution, priming MSCs sensitive to signaling, thus stimulating differentiation into a specific cell lineage, may improve the differentiation yield. To demonstrate this, priming MSCs were produced by using a gelatin matrix for the isolation of primary MSCs from bone-marrow-derived primary cells. Subsequently, cellular characteristics and sensitivity to specific differentiation signals were analyzed at passage five. Compared to non-priming MSCs, priming MSCs showed no significant differences in cellular characteristics, but demonstrated a significant increase in sensitivity to neurogenic differentiation signals. These results demonstrate that generation of priming MSCs by specific extracellular signaling increases the rate of differentiation into a cell-specific lineage.

  18. Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system.

    PubMed

    Ram, Senthoor K; Raval, Keyur; JagadeeshBabu, P E

    2015-01-01

    Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

  19. Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions.

    PubMed

    Voulgaris, Ioannis; Finka, Gary; Uden, Mark; Hoare, Mike

    2015-10-01

    The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1 kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation was with the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli.

  20. Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions.

    PubMed

    Voulgaris, Ioannis; Finka, Gary; Uden, Mark; Hoare, Mike

    2015-10-01

    The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1 kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation was with the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli. PMID:26184976

  1. Endoplasmic reticulum is a key organella in bradykinin-triggered ATP release from cultured smooth muscle cells.

    PubMed

    Zhao, Yumei; Migita, Keisuke; Sato, Chiemi; Usune, Sadaharu; Iwamoto, Takahiro; Katsuragi, Takeshi

    2007-09-01

    ATP has broad functions as an autocrine/paracrine molecule. The mode of ATP release and its intracellular source, however, are little understood. Here we show that bradykinin via B(2)-receptor stimulation induces the extracellular release of ATP via the inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]-signaling pathway in cultured taenia coli smooth muscle cells. It was found that bradykinin also increased the production of Ins(1,4,5)P(3) and 2-APB-inhibitable [Ca(2+)](i). The evoked release of ATP was suppressed by the Ca(2+)-channel blockers, nifedipine, and verapamil. Moreover, the extracellular release of ATP was elicited by photoliberation of Ins(1,4,5)P(3). Bradykinin caused a quick and transient accumulation of intracellular ATP from cells treated with 1% perchloric acid solution (PCA), but not with the cell lysis buffer. Peak accumulation was prevented by 2-APB and thapsigargin, but not by nifedipine or verapamil, inhibitors of extracellular release of ATP. These findings suggest that bradykinin elicits the extracellular release of ATP that is mediated by the Ins(1,4,5)P(3)-induced Ca(2+) signaling and, finally, leads to a Ca(2+)-dependent export of ATP from the cells. Furthermore, the bradykinin-induced transient accumulation of ATP in the cells treated with PCA may imply a possible release of ATP from the endoplasmic reticulum.

  2. Acidic extracellular pH promotes prostate cancer bone metastasis by enhancing PC-3 stem cell characteristics, cell invasiveness and VEGF-induced vasculogenesis of BM-EPCs.

    PubMed

    Huang, Sheng; Tang, Yubo; Peng, Xinsheng; Cai, Xingdong; Wa, Qingde; Ren, Dong; Li, Qiji; Luo, Jiaquan; Li, Liangping; Zou, Xuenong; Huang, Shuai

    2016-10-01

    Bone metastasis is a main cause of cancer-related mortality in patients with advanced prostate cancer. Emerging evidence suggests that the acidic extracellular microenvironment plays significant roles in the growth and metastasis of tumors. However, the effects of acidity on bone metastasis of PCa remain undefined. In the present study, PC-3 cells were cultured in acidic medium (AM; pH 6.5) or neutral medium (NM; pH 7.4), aiming to investigate the effects and possible mechanisms of acidic extracellular microenvironment in bone metastasis of PCa. Our results showed that AM can promote spheroid and colony formations, cell viability and expression of stem cell characteristic-related markers in PC-3 cells. Moreover, AM stimulates MMP-9 secretion and promotes invasiveness of PC-3 cells, and these effects can be inhibited by blocking of MMP-9. Furthermore, AM stimulates VEGF secretion of PC-3 and AM conditioned medium (CMAM) promotes vasculogenesis of BM-EPCs by increasing cell viability, migration, tube formation, which involved activating the phosphorylation of VEGFR-2, Akt and P38, when pH of NM conditioned medium (CMNM) was modulated the same as AM conditioned medium (CMAM). Further studies have shown that CMNM induced vasculogenesis of BM-EPCs can be inhibited by the inhibition of VEGFR2 with DMH4. These findings suggest that acidic extracellular microenvironment may have the potential to modulate prostate cancer bone metastasis by enhancing PC-3 stem cell characteristics, cell invasiveness and VEGF-induced vasculogenesis of BM-EPCs. Improved anticancer strategies should be designed to selectively target acidic tumor microenvironment.

  3. HRG-1 enhances cancer cell invasive potential and couples glucose metabolism to cytosolic/extracellular pH gradient regulation by the vacuolar-H(+) ATPase.

    PubMed

    Fogarty, F M; O'Keeffe, J; Zhadanov, A; Papkovsky, D; Ayllon, V; O'Connor, R

    2014-09-18

    Haeme-responsive gene (HRG)-1 encodes a 16-kDa transmembrane protein that is induced by insulin-like growth factor-1 (IGF-1) and associates with the vacuolar-(H(+)) ATPase (V-ATPase). We previously reported that HRG-1 is essential for V-ATPase activity in endosomal acidification and receptor trafficking. Here, we show that in highly invasive and migratory cancer cell lines, HRG-1 and the V-ATPase are co-expressed at the plasma membrane, whereas in less invasive cell lines and non-transformed cells HRG-1 over-expression remains confined to intracellular compartments. Stable suppression of HRG-1 in invasive breast cancer MDA-MB-231 cells decreases extracellular pH, cell growth, migration and invasion. Ectopic expression of HRG-1 in non-invasive MCF-7 cells enhances V-ATPase activity, lowers the extracellular pH and increases the pH-dependent activity of MMP2 and MMP9 matrix metalloproteinases. HRG-1 enhances trafficking of the glucose transporter-1 (GLUT-1) with a concomitant increase in glucose uptake and lactate production. HRG-1 also promotes trafficking of the insulin-like growth factor I receptor (IGF-1R), β1-integrin and IGF-1 signalling. Taken together, our findings indicate that HRG-1 expression at the plasma membrane enhances V-ATPase activity, drives glycolytic flux and facilitates cancer cell growth, migration and invasion. Thus, HRG-1 may represent a novel target for selectively disrupting V-ATPase activity and the metastatic potential of cancer cells.

  4. Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

    PubMed Central

    Arcangeli, Sara; Nasti, Annamaria Assunta; Giordano, Antonio; Amoroso, Salvatore

    2012-01-01

    Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production. PMID:22479505

  5. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease. PMID:26195825

  6. ATP as a cotransmitter in the autonomic nervous system.

    PubMed

    Kennedy, Charles

    2015-09-01

    The role of adenosine 5'-triphosphate (ATP) as a major intracellular energy source is well-established. In addition, ATP and related nucleotides have widespread extracellular actions via the ionotropic P2X (ligand-gated cation channels) and metabotropic P2Y (G protein-coupled) receptors. Numerous experimental techniques, including myography, electrophysiology and biochemical measurement of neurotransmitter release, have been used to show that ATP has several major roles as a neurotransmitter in peripheral nerves. When released from enteric nerves of the gastrointestinal tract it acts as an inhibitory neurotransmitter, mediating descending muscle relaxation during peristalsis. ATP is also an excitatory cotransmitter in autonomic nerves; 1) It is costored with noradrenaline in synaptic vesicles in postganglionic sympathetic nerves innervating smooth muscle preparations, such as the vas deferens and most arteries. When coreleased with noradrenaline, ATP acts at postjunctional P2X1 receptors to evoke depolarisation, Ca(2+) influx, Ca(2+) sensitisation and contraction. 2) ATP is also coreleased with acetylcholine from postganglionic parasympathetic nerves innervating the urinary bladder and again acts at postjunctional P2X1 receptors, and possibly also a P2X1+4 heteromer, to elicit smooth muscle contraction. In both cases the neurotransmitter actions of ATP are terminated by dephosphorylation by extracellular, membrane-bound enzymes and soluble nucleotidases released from postganglionic nerves. There are indications of an increased contribution of ATP to control of blood pressure in hypertension, but further research is needed to clarify this possibility. More promising is the upregulation of P2X receptors in dysfunctional bladder, including interstitial cystitis, idiopathic detrusor instability and overactive bladder syndrome. Consequently, these roles of ATP are of great therapeutic interest and are increasingly being targeted by pharmaceutical companies.

  7. ATP stimulates pannexin 1 internalization to endosomal compartments.

    PubMed

    Boyce, Andrew K J; Kim, Michelle S; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2015-09-15

    The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease.

  8. Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury

    PubMed Central

    Saha, Subhrajit; Aranda, Evelyn; Hayakawa, Yoku; Bhanja, Payel; Atay, Safinur; Brodin, N Patrik; Li, Jiufeng; Asfaha, Samuel; Liu, Laibin; Tailor, Yagnesh; Zhang, Jinghang; Godwin, Andrew K.; Tome, Wolfgang A.; Wang, Timothy C.; Guha, Chandan; Pollard, Jeffrey W.

    2016-01-01

    WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation. PMID:27734833

  9. Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment.

    PubMed

    He, Ningning; Xu, Yang; Du, Wei; Qi, Xin; Liang, Lu; Wang, Yuebing; Feng, Guowei; Fan, Yan; Han, Zhongchao; Kong, Deling; Cheng, Zhen; Wu, Joseph C; He, Zuoxiang; Li, Zongjin

    2015-01-01

    The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes' expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

  10. An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices

    PubMed Central

    Jung, Jangwook P.; Hu, Dongjian; Domian, Ibrahim J.; Ogle, Brenda M.

    2015-01-01

    The extracellular matrix (ECM) impacts stem cell differentiation, but identifying formulations supportive of differentiation is challenging in 3D models. Prior efforts involving combinatorial ECM arrays seemed intuitively advantageous. We propose an alternative that suggests reducing sample size and technological burden can be beneficial and accessible when coupled to design of experiments approaches. We predict optimized ECM formulations could augment differentiation of cardiomyocytes derived in vitro. We employed native chemical ligation to polymerize 3D poly (ethylene glycol) hydrogels under mild conditions while entrapping various combinations of ECM and murine induced pluripotent stem cells. Systematic optimization for cardiomyocyte differentiation yielded a predicted solution of 61%, 24%, and 15% of collagen type I, laminin-111, and fibronectin, respectively. This solution was confirmed by increased numbers of cardiac troponin T, α-myosin heavy chain and α-sarcomeric actinin-expressing cells relative to suboptimum solutions. Cardiomyocytes of composites exhibited connexin43 expression, appropriate contractile kinetics and intracellular calcium handling. Further, adding a modulator of adhesion, thrombospondin-1, abrogated cardiomyocyte differentiation. Thus, the integrated biomaterial platform statistically identified an ECM formulation best supportive of cardiomyocyte differentiation. In future, this formulation could be coupled with biochemical stimulation to improve functional maturation of cardiomyocytes derived in vitro or transplanted in vivo. PMID:26687770

  11. Electrolyte Cations Binding with Extracellular Polymeric Substances Enhanced Microcystis Aggregation: Implication for Microcystis Bloom Formation in Eutrophic Freshwater Lakes.

    PubMed

    Xu, Huacheng; Lv, Hua; Liu, Xin; Wang, Peifang; Jiang, Helong

    2016-09-01

    The hydrodynamic and structural properties of Microcystis extracellular polymeric substances (EPS) in electrolytes with different valences and ionic strengths were investigated via using dynamic light scattering, the fluorescence excitation emission matrix coupled with parallel factor (EEM-PARAFAC) analysis, two-dimensional correlation spectroscopy (2D-COS), and cryogenic transmission electron microscopy (Cryo-TEM). The hydrodynamic diameters of EPS colloids exhibited no variation for monovalent NaCl but a substantial increase for divalent CaCl2 and MgCl2. However, the negative electrophoretic mobilities for all complexes indicated that charge neutralization would not be the main mechanism for EPS aggregation. Application of EEM-PARAFAC and 2D-Fourier transform infrared (FTIR)-COS revealed obvious electrolyte binding potential with both fluorescent phenolic and aromatic compounds and nonfluorescent polysaccharides. The complexation model showed that divalent Ca(2+) and Mg(2+) exhibited a strong binding capability with phenolic -OH, aromatic C═C, and polysaccharide C-O groups, while the monovalent electrolyte exhibited negligible association with these groups. Such a strong complexation can bridge each individual biomolecule together to form EPS aggregates and Microcystis colonies, as supported by in situ Cryo-TEM and light microscope observation, respectively. Given the increased concentration in natural ecosystems, electrolyte cations, especially divalent cations, would play increased roles in Microcystis bloom formation and thus should be considered. PMID:27502019

  12. Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia

    PubMed Central

    Moorthy, Anandi Narayana; Rai, Prashant; Jiao, Huipeng; Wang, Shi; Tan, Kong Bing; Qin, Liang; Watanabe, Hiroshi; Zhang, Yongliang; Teluguakula, Narasaraju; Chow, Vincent Tak Kwong

    2016-01-01

    Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia. PMID:27034012

  13. Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks.

    PubMed

    Tutak, Wojtek; Park, Ki Ho; Vasilov, Anatoly; Starovoytov, Valentin; Fanchini, Giovanni; Cai, Shi-Qing; Partridge, Nicola C; Sesti, Federico; Chhowalla, Manish

    2009-06-24

    A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

  14. Pannexin1-Mediated ATP Release Provides Signal Transmission Between Neuro2A Cells

    PubMed Central

    Iglesias, Rodolfo M.

    2013-01-01

    Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell–cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from −60 to +100 mV; threshold was correlated with extracellular K+, so that at 10 mM K+, channels began to open at −30 mV. Evaluation of cell–cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10–20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 µM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 µM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K+ provides a mechanism for enhancement of ATP release under pathological conditions. PMID:22359052

  15. Interaction of TGA@CdTe Quantum Dots with an Extracellular Matrix of Haematococcus pluvialis Microalgae Detected Using Surface-Enhanced Raman Spectroscopy (SERS).

    PubMed

    Cepeda-Pérez, Elisa; Aguilar-Hernández, Iris; López-Luke, Tzarara; Piazza, Valeria; Carriles, Ramón; Ornelas-Soto, Nancy; de la Rosa, Elder

    2016-09-01

    The present study reports the localization and interaction of thioglycolic acid (TGA) capped CdTe quantum dots (TGA@CdTe QDs) within the extracellular matrix (ECM) of Haematococcus pluvialis (Chlorophyceae) microalgae (HPM) after an incubation period of 5 min. Changes in the Raman spectrum of HPM induced by the adsorption of the TGA@CdTe QDs are successfully found by using naked gold anisotropic structures as nano-sensors for surface-enhanced Raman scattering (SERS effect). Raman spectroscopy results show that TGA@CdTe QDs interact with the biomolecules present in the ECM. Sample preparation and characterization by complementary techniques such as confocal and electron microscopy are also used to confirm the presence and localization of the nanoparticles in the algae. This research shows new evidence on early accumulation of QDs in plant cells and would further improve our understanding about their environmental impact. PMID:27381350

  16. Interaction of TGA@CdTe Quantum Dots with an Extracellular Matrix of Haematococcus pluvialis Microalgae Detected Using Surface-Enhanced Raman Spectroscopy (SERS).

    PubMed

    Cepeda-Pérez, Elisa; Aguilar-Hernández, Iris; López-Luke, Tzarara; Piazza, Valeria; Carriles, Ramón; Ornelas-Soto, Nancy; de la Rosa, Elder

    2016-09-01

    The present study reports the localization and interaction of thioglycolic acid (TGA) capped CdTe quantum dots (TGA@CdTe QDs) within the extracellular matrix (ECM) of Haematococcus pluvialis (Chlorophyceae) microalgae (HPM) after an incubation period of 5 min. Changes in the Raman spectrum of HPM induced by the adsorption of the TGA@CdTe QDs are successfully found by using naked gold anisotropic structures as nano-sensors for surface-enhanced Raman scattering (SERS effect). Raman spectroscopy results show that TGA@CdTe QDs interact with the biomolecules present in the ECM. Sample preparation and characterization by complementary techniques such as confocal and electron microscopy are also used to confirm the presence and localization of the nanoparticles in the algae. This research shows new evidence on early accumulation of QDs in plant cells and would further improve our understanding about their environmental impact.

  17. Measurement of ATP-Induced Membrane Potential Changes in IVD cells

    PubMed Central

    Gonzales, Silvia; Rodriguez, Brittany; Barrera, Carlos; Huang, Chun-Yuh Charles

    2014-01-01

    Extracellular adenosine-5'-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus (AF) cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X4 purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonate (PPADS), a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors. PMID:25386223

  18. Enhancing T-DNA Transfer Efficiency in Barley (Hordeum vulgare L.) Cells Using Extracellular Cellulose and Lectin.

    PubMed

    Gürel, Filiz; Uçarlı, Cüneyt; Tufan, Feyza; Kalaskar, Deepak M

    2015-06-01

    A major limitation of transforming barley tissues by Agrobacterium tumefaciens is the low frequency of T-DNA transfer due to recalcitrance of barley as a host. The effect of extracellular cellulose and lectin on Agrobacterium transformation efficiency was investigated in this study. Barley callus cultures were transformed with the AGL1 strain containing the vector pBI121 in the presence of 10 mg mL(-1) cellulose or 0.001, 0.05 and 0.1 mg mL(-1) lectin. Addition of cellulose significantly (P ≤ 0.05) increased the number of GUS spots by 50 % compared to standard conditions in the presence of only 200 μM acetosyringone (AS). Frequency of G418-resistant aggregates on the surfaces of callus cultures was 29 and 71.5 %, following AS and AS + cellulose treatments, respectively, after 4 weeks of selection. Presence of 0.05 or 0.1 mg mL(-1) lectin also increased the number of GUS spots and frequency of G418-resistant cells in the selection period, but the increase in blue spots was not significant. We examined the effect of lectin and cellulose on bacterial attachment to callus tissues. Both cellulose and lectin were found to have a significant positive effect on the numbers of bacteria attached to barley callus. Epifluorescence microscopy revealed that Agrobacterium cells had accumulated in the scaffolds of irregular fibrous cellulose with a mean particle size of 200 μm. Expression of nptII in transformed callus lines confirmed the stable transformation of the gene. Our study showed for the first time the binding of Agrobacterium cells to fibrous cellulose and also demonstrated how polysaccharides and glycoproteins can be used to improve T-DNA transfer in monocotyledon transformation procedures.

  19. Degradation of slime extracellular polymeric substances and inhibited sludge flocs destruction contribute to sludge dewaterability enhancement during fungal treatment of sludge using filamentous fungus Mucor sp. GY-1.

    PubMed

    Wang, Zhenyu; Zheng, Guanyu; Zhou, Lixiang

    2015-09-01

    Mechanisms responsible for the sludge dewaterability enhanced by filamentous fungi during fungal treatment of sludge were investigated in the present study. The filamentous fungus Mucor sp. GY-1, isolated from waste activated sludge, enhanced sludge dewaterability by 82.1% to achieve the lowest value of normalized sludge specific resistance to filtration (SRF), 8.18 × 10(10) m · L/kg · g-TSS. During the fungal treatment of sludge, 57.8% of slime extracellular polymeric substances (EPS) and 51.1% of polysaccharide in slime EPS were degraded, respectively, by Mucor sp. GY-1, contributing to the improvement of sludge dewaterability. Slime EPS is much more available for Mucor sp. GY-1 than either LB-EPS or TB-EPS that bound with microbial cells. In addition, filamentous fungus Mucor sp. GY-1 entrapped small sludge particles and inhibited the destruction of sludge flocs larger than 100 μm, thus enhancing sludge dewaterability, during fungal treatment of sludge using Mucor sp. GY-1.

  20. Degradation of slime extracellular polymeric substances and inhibited sludge flocs destruction contribute to sludge dewaterability enhancement during fungal treatment of sludge using filamentous fungus Mucor sp. GY-1.

    PubMed

    Wang, Zhenyu; Zheng, Guanyu; Zhou, Lixiang

    2015-09-01

    Mechanisms responsible for the sludge dewaterability enhanced by filamentous fungi during fungal treatment of sludge were investigated in the present study. The filamentous fungus Mucor sp. GY-1, isolated from waste activated sludge, enhanced sludge dewaterability by 82.1% to achieve the lowest value of normalized sludge specific resistance to filtration (SRF), 8.18 × 10(10) m · L/kg · g-TSS. During the fungal treatment of sludge, 57.8% of slime extracellular polymeric substances (EPS) and 51.1% of polysaccharide in slime EPS were degraded, respectively, by Mucor sp. GY-1, contributing to the improvement of sludge dewaterability. Slime EPS is much more available for Mucor sp. GY-1 than either LB-EPS or TB-EPS that bound with microbial cells. In addition, filamentous fungus Mucor sp. GY-1 entrapped small sludge particles and inhibited the destruction of sludge flocs larger than 100 μm, thus enhancing sludge dewaterability, during fungal treatment of sludge using Mucor sp. GY-1. PMID:26086084

  1. Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B*

    PubMed Central

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2012-01-01

    ATP7A and ATP7B are copper-transporting P1B-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis. PMID:22130675

  2. K(ATP)-channel-dependent regulation of catecholaminergic neurons controls BAT sympathetic nerve activity and energy homeostasis.

    PubMed

    Tovar, Sulay; Paeger, Lars; Hess, Simon; Morgan, Donald A; Hausen, A Christine; Brönneke, Hella S; Hampel, Brigitte; Ackermann, P Justus; Evers, Nadine; Büning, Hildegard; Wunderlich, F Thomas; Rahmouni, Kamal; Kloppenburg, Peter; Brüning, Jens C

    2013-09-01

    Brown adipose tissue (BAT) is a critical regulator of glucose, lipid, and energy homeostasis, and its activity is tightly controlled by the sympathetic nervous system. However, the mechanisms underlying CNS-dependent control of BAT sympathetic nerve activity (SNA) are only partly understood. Here, we demonstrate that catecholaminergic neurons in the locus coeruleus (LC) adapt their firing frequency to extracellular glucose concentrations in a K(ATP)-channel-dependent manner. Inhibiting K(ATP)-channel-dependent control of neuronal activity via the expression of a variant K(ATP) channel in tyrosine-hydroxylase-expressing neurons and in neurons of the LC enhances diet-induced obesity in mice. Obesity results from decreased energy expenditure, lower steady-state BAT SNA, and an attenuated ability of centrally applied glucose to activate BAT SNA. This impairs the thermogenic transcriptional program of BAT. Collectively, our data reveal a role of K(ATP)-channel-dependent neuronal excitability in catecholaminergic neurons in maintaining thermogenic BAT sympathetic tone and energy homeostasis.

  3. Impaired mitochondrial Ca{sup 2+} homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

    SciTech Connect

    Kleist-Retzow, Juergen-Christoph von ||. E-mail: juergen-christoph.vonkleist@uk-koeln.de; Hue-Tran Hornig-Do; Schauen, Matthias; Eckertz, Sabrina; Tuan Anh Duong Dinh; Stassen, Frank; Lottmann, Nadine; Bust, Maria; Galunska, Bistra; Wielckens, Klaus; Hein, Wolfgang; Beuth, Joseph; Braun, Jan-Matthias; Fischer, Juergen H.; Ganitkevich, Vladimir Y. |; Maniura-Weber, Katharina; Wiesner, Rudolf J. |

    2007-08-15

    Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca{sup 2+} homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNA{sup Leu(UUR)}, as well as Rho{sup 0} cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.

  4. ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

    PubMed

    Ueda, T; Nakata, Y; Yamasaki, N; Oda, H; Sentani, K; Kanai, A; Onishi, N; Ikeda, K; Sera, Y; Honda, Z-I; Tanaka, K; Sata, M; Ogawa, S; Yasui, W; Saya, H; Takita, J; Honda, H

    2016-08-25

    Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors. PMID:26829053

  5. Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand.

    PubMed

    Yaniv, Yael; Spurgeon, Harold A; Ziman, Bruce D; Lyashkov, Alexey E; Lakatta, Edward G

    2013-06-01

    The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O₂ consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O₂ consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

  6. Extracellular Methemoglobin Mediated Early ROS Spike Triggers Osmotic Fragility and RBC Destruction: An Insight into the Enhanced Hemolysis During Malaria.

    PubMed

    Balaji, S N; Trivedi, Vishal

    2012-04-01

    Malaria infection is known to cause severe hemolysis due to production of abnormal RBCs and enhanced RBC destruction through apoptosis. Infected RBC lysis exposes uninfected RBC to the large amount of pro-oxidant molecules such as methemoglobin. Methemoglobin (MetHb) exposure dose dependently makes RBCs susceptible to osmotic stress and causes hemolysis. MetHb mediated oxidative stress in RBC correlated well with osmotic fragility and hemolysis. Interestingly, a reactive oxygen species (ROS) spike at 15 min was responsible for the observed effects on RBC cells. Two natural antioxidants N-acetyl cysteine and mannitol protected the RBC from MetHb-mediated defects, which clearly indicated involvement of oxidative stress in the process. MetHb due to its pseudo-peroxidase activity produces ROS in the external microenvironment. Therefore, classical peroxidase inhibitors were tested to probe peroxidase activity mediated ROS production with defects in RBCs. Clotrimazole (CLT), which irreversibly inactivates the MetHb (CLT-MetHb) and abolishes peroxidase activity, did not produce significant ROS outside RBC and was inefficient to cause osmotic fragility and hemolysis. Hence, initiating a chain reaction, MetHb released from ruptured RBC produces significant ROS in the external microenvironment to make RBC membrane leaky and enhanced hemolysis. Together data presented in the current work explored the role of MetHb in accelerated humorless during malaria which could be responsible for severe outcomes of pathological disorders.

  7. Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca{sup 2+}

    SciTech Connect

    Hashimoto, Ryota; Katoh, Youichi; Nakamura, Kyoko; Itoh, Seigo; Iesaki, Takafumi; Daida, Hiroyuki; Nakazato, Yuji; Okada, Takao

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} enhances adipocyte accumulation in the presence of adipogenic inducers. Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} enhances both proliferation and adipocyte differentiation in BMSCs. Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub o} in BMSCs. Black-Right-Pointing-Pointer An intracellular Ca{sup 2+} chelator suppresses the enhancement in adipocyte accumulation. Black-Right-Pointing-Pointer Controlling [Ca{sup 2+}]{sub o} may govern the balance of adipocyte and osteoblast development. -- Abstract: The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca{sup 2+} levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca{sup 2+}]{sub o} on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca{sup 2+}]{sub o} enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub i}. An intracellular Ca{sup 2+} chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca{sup 2+}]{sub o} in BMSCs. These data suggest a new role for extracellular Ca{sup 2+} in the bone marrow stroma: increased [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub i} levels, which in turn enhances the accumulation of

  8. Compartmentalized ATP synthesis in skeletal muscle triads.

    PubMed

    Han, J W; Thieleczek, R; Varsányi, M; Heilmeyer, L M

    1992-01-21

    Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads. PMID:1731894

  9. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro

    PubMed Central

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  10. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro.

    PubMed

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-09-20

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson's disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe(2+)-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment.

  11. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro.

    PubMed

    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson's disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe(2+)-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  12. The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis

    PubMed Central

    Koh, Eun-Ik; Hung, Chia S.

    2016-01-01

    The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis. In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 106-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs. PMID:26883590

  13. The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis.

    PubMed

    Koh, Eun-Ik; Hung, Chia S; Henderson, Jeffrey P

    2016-05-01

    The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 10(6)-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs. PMID:26883590

  14. The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.

    PubMed

    Sasser, Terry L; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A

    2013-06-21

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.

  15. ATP-induced cardioprotection against myocardial ischemia/reperfusion injury is mediated through the RISK pathway

    PubMed Central

    Lian, Zhe-Xun; Wang, Fang; Fu, Jun-Hua; Chen, Zuo-Yuan; Xin, Hui; Yao, Ru-Yong

    2016-01-01

    The aim of the present study was to examine the post-infarct acute effect of adenosine-5′-triphosphate (ATP) on myocardial infarction (MI) size as well as its precise molecular mechanism. Sixty New Zealand white male rabbits were exposed to 40 min of ischemia followed by 180 min of reperfusion. The rabbits were intravenously administered 3 mg/kg of ATP (ATP group) or saline (control group) immediately after reperfusion and maintained throughout the first 30 min. The wortmannin+ATP, PD-98059+ATP, and 5-hydroxydecanoic acid (5-HD) sodium salt+ATP groups were separately injected with wortmannin (0.6 mg/kg), PD-98059 (0.3 mg/kg), and 5-HD (5 mg/kg) 5 min prior to ATP administration. MI size was calculated as the percentage of the risk area in the left ventricle. Myocardial apoptosis was determined using a TUNEL assay. Western blot analysis was performed to examine the levels of protein kinase B (Akt)/p-Akt and extracellular signal-regulated kinase (ERK)/p-ERK in the ischemic myocardium, 180 min after reperfusion. The infarct size was significantly smaller in the ATP group than in the control group (p<0.05). The infarct size-reducing effect of ATP was completely blocked by wortmannin, PD-98059 and 5-HD. Compared with the control group, cardiomyocyte apoptosis was significantly reduced in the ATP group, while this did not occur in the wortmannin+ATP, PD-98059+ATP and 5-HD+ATP groups. Western blot analysis revealed a higher myocardial expression of p-Akt and p-ERK 180 min following reperfusion in the ATP versus the control group. In conclusion, cardioprotection by postischemic ATP administration is mediated through activation of the reperfusion injury salvage kinase (RISK) pathway and opening of the mitochondrial ATP-dependent potassium channels. PMID:27698693

  16. ATP-induced cardioprotection against myocardial ischemia/reperfusion injury is mediated through the RISK pathway

    PubMed Central

    Lian, Zhe-Xun; Wang, Fang; Fu, Jun-Hua; Chen, Zuo-Yuan; Xin, Hui; Yao, Ru-Yong

    2016-01-01

    The aim of the present study was to examine the post-infarct acute effect of adenosine-5′-triphosphate (ATP) on myocardial infarction (MI) size as well as its precise molecular mechanism. Sixty New Zealand white male rabbits were exposed to 40 min of ischemia followed by 180 min of reperfusion. The rabbits were intravenously administered 3 mg/kg of ATP (ATP group) or saline (control group) immediately after reperfusion and maintained throughout the first 30 min. The wortmannin+ATP, PD-98059+ATP, and 5-hydroxydecanoic acid (5-HD) sodium salt+ATP groups were separately injected with wortmannin (0.6 mg/kg), PD-98059 (0.3 mg/kg), and 5-HD (5 mg/kg) 5 min prior to ATP administration. MI size was calculated as the percentage of the risk area in the left ventricle. Myocardial apoptosis was determined using a TUNEL assay. Western blot analysis was performed to examine the levels of protein kinase B (Akt)/p-Akt and extracellular signal-regulated kinase (ERK)/p-ERK in the ischemic myocardium, 180 min after reperfusion. The infarct size was significantly smaller in the ATP group than in the control group (p<0.05). The infarct size-reducing effect of ATP was completely blocked by wortmannin, PD-98059 and 5-HD. Compared with the control group, cardiomyocyte apoptosis was significantly reduced in the ATP group, while this did not occur in the wortmannin+ATP, PD-98059+ATP and 5-HD+ATP groups. Western blot analysis revealed a higher myocardial expression of p-Akt and p-ERK 180 min following reperfusion in the ATP versus the control group. In conclusion, cardioprotection by postischemic ATP administration is mediated through activation of the reperfusion injury salvage kinase (RISK) pathway and opening of the mitochondrial ATP-dependent potassium channels.

  17. Extracellular nucleotides regulate cellular functions of podocytes in culture.

    PubMed

    Fischer, K G; Saueressig, U; Jacobshagen, C; Wichelmann, A; Pavenstädt, H

    2001-12-01

    Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors.

  18. Extracellular nucleotides regulate cellular functions of podocytes in culture.

    PubMed

    Fischer, K G; Saueressig, U; Jacobshagen, C; Wichelmann, A; Pavenstädt, H

    2001-12-01

    Extracellular nucleotides are assumed to be important regulators of glomerular functions. This study characterizes purinergic receptors in podocytes. The effects of purinergic agonists on electrophysiological properties and the intracellular free Ca(2+) concentration of differentiated podocytes were examined with the patch-clamp and fura 2 fluorescence techniques. mRNA expression of purinergic receptors was investigated by RT-PCR. Purinergic agonists depolarized podocytes. Purinergic agonists similarly increased intracellular free Ca(2+) concentration of podocytes. The rank order of potency of various nucleotides on membrane voltage and free cytosolic calcium concentration was UTP approximately UDP > [adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S)] > ATP > 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) > 2'- and 3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) > ADP-beta-S. alpha,beta-Me-ATP was without effect. In the presence of UTP, BzATP did not cause an additional depolarization of podocytes. Incubation of cells with ATP or BzATP did not induce lactate dehydrogenase release. In RT-PCR studies, mRNAs of the P2Y(1), P2Y(2), P2Y(6), and P2X(7) receptors were detected within glomeruli and podocytes. The data indicate that extracellular nucleotides modulate podocyte function mainly by an activation of both P2Y(2) and P2Y(6) receptors. PMID:11704558

  19. KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion

    PubMed Central

    Deeney, Jude T.; Corkey, Barbara E.

    2015-01-01

    Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS–1 cells. Taking advantage of hemicannels’opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion. PMID:26444014

  20. 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?"

  1. ATP Release from Vascular Endothelia Occurs Across Cx43 Hemichannels and Is Attenuated during Hypoxia

    PubMed Central

    Zug, Stephanie; El Kasmi, Karim C.; Eltzschig, Holger K.

    2008-01-01

    Background Extracellular ATP is an important signaling molecule for vascular adaptation to limited oxygen availability (hypoxia). Here, we pursued the contribution of vascular endothelia to extracellular ATP release under hypoxic conditions. Methodology, Principal Findings We gained first insight from studying ATP release from endothelia (HMEC-1) pre-exposed to hypoxia. Surprisingly, we found that ATP release was significantly attenuated following hypoxia exposure (2% oxygen, 22±3% after 48 h). In contrast, intracellular ATP was unchanged. Similarly, lactate-dehydrogenase release into the supernatants was similar between normoxic or hypoxic endothelia, suggesting that differences in lytic ATP release between normoxia or hypoxia are minimal. Next, we used pharmacological strategies to study potential mechanisms for endothelial-dependent ATP release (eg, verapamil, dipyridamole, 18-alpha-glycyrrhetinic acid, anandamide, connexin-mimetic peptides). These studies revealed that endothelial ATP release occurs – at least in part - through connexin 43 (Cx43) hemichannels. A real-time RT-PCR screen of endothelial connexin expression showed selective repression of Cx43 transcript and additional studies confirmed time-dependent Cx43 mRNA, total and surface protein repression during hypoxia. In addition, hypoxia resulted in Cx43-serine368 phosphorylation, which is known to switch Cx43 hemi-channels from an open to a closed state. Conclusions/Significance Taken together, these studies implicate endothelial Cx43 in hypoxia-associated repression of endothelial ATP release. PMID:18665255

  2. 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

  3. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  4. Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury.

    PubMed Central

    Ogawa, K.; Suzuki, J.; Mukai, H.; Mori, M.

    1986-01-01

    Immunohistochemical investigations were carried out on the properties of the cells and extracellular matrix (ECM) in focal hepatic injuries. A liquid nitrogen-cooled syringe needle was thrust into the rat liver. Necrotic areas became permeated with plasma within 24-hour period. Areas became strongly positive for fibronectin and were infiltrated with inflammatory cells positive for lysozyme. By the third day, Ito cells were proliferated in the peripheral portions of the damaged areas. These Ito cells showed enhanced immunostaining for desmin and actin but were negative for lysozyme. Interstitial fibers which were immunochemically positive for Types I and IV collagens, laminin, and fibronectin, began to increase from Day 3. They appeared on the rim of the hepatocytes adjacent to the damaged areas and extended into the injured regions with the Ito cells. An increase in basal laminas associated with capillaries and bile ducts also increased with a 1-day delay. The damaged areas were replaced by granulation tissue by Day 5. A rapid diminution then occurred in the granulation tissue, and normal hepatic tissue was restored in 7-10 days. These observations demonstrate that ECM changed in a sequential manner and then finally disappeared from the damaged site within 10 days. Although various cells, including parenchymal cells, macrophages, endothelial cells, and cholangiolar cells contributed to the healing of the damaged area, Ito cells, which exhibit unique phenotypic changes, presumably had a major role in the process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3799820

  5. Characterization of the extracellular biodemulsifiers secreted by Bacillus cereus LH-6 and the enhancement of demulsifying efficiency by optimizing the cultivation conditions.

    PubMed

    Hou, Ning; Feng, Fengzhao; Shi, Yan; Cao, Huiming; Li, Chunyan; Cao, Zhi; Cheng, Yi

    2014-09-01

    A highly efficient demulsifying strain, LH-6, was isolated from petroleum-contaminated soil and identified as Bacillus cereus by 16S rDNA gene analysis. It achieved 95.61 and 95.40 % demulsifying ratios within 12 h for water-in-oil (W/O) and oil-in-water (O/W) model emulsions, respectively. Fourier transform infrared spectroscopy (FT-IR) and thin-layer chromatography (TLC) detections indicated that the LH-6's extracellular biodemulsifiers were different types of lipopeptides for the W/O and O/W emulsions. Optimization of the culture medium composition was conducted to improve the biosynthesis and demulsifying efficiency of the biodemulsifier. The optimal carbon source was liquid paraffin, while waste frying oil could also be an alternative carbon source. The optimal nitrogen sources were ammonium sulfate and yeast extract. To further enhance the biodemulsifier efficiency, the optimal cultivation conditions were determined using response surface methodology (RSM) based on central composite rotation design (CCRD). Using the optimized cultivation conditions, the demulsifying ratios increased to 98.23 and 97.65 % for the W/O and O/W model emulsions, respectively.

  6. Reduction of inflammatory responses and enhancement of extracellular matrix formation by vanillin-incorporated poly(lactic-co-glycolic acid) scaffolds.

    PubMed

    Lee, Yujung; Kwon, Jeongil; Khang, Gilson; Lee, Dongwon

    2012-10-01

    Vanillin is one of the major components of vanilla, a commonly used flavoring agent and preservative and is known to exert potent antioxidant and anti-inflammatory activities. In this work, vanillin-incorporated poly(lactic-co-glycolic acid) (PLGA) films and scaffolds were fabricated to evaluate the effects of vanillin on the inflammatory responses and extracellular matrix (ECM) formation in vitro and in vivo. The incorporation of vanillin to PLGA films induced hydrophilic nature, resulting in the higher cell attachment and proliferation than the pure PLGA film. Vanillin also reduced the generation of reactive oxygen species (ROS) in cells cultured on the pure PLGA film and significantly inhibited the PLGA-induced inflammatory responses in vivo, evidenced by the reduced accumulation of inflammatory cells and thinner fibrous capsules. The effects of vanillin on the ECM formation were evaluated using annulus fibrous (AF) cell-seeded porous PLGA/vanillin scaffolds. PLGA/vanillin scaffolds elicited the more production of glycosaminoglycan and collagen than the pure PLGA scaffold, in a concentration-dependent manner. Based on the low level of inflammatory responses and enhanced ECM formation, vanillin-incorporated PLGA constructs make them promising candidates in the future biomedical applications.

  7. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  8. A Tissue-Engineered Chondrocyte Cell Sheet Induces Extracellular Matrix Modification to Enhance Ventricular Biomechanics and Attenuate Myocardial Stiffness in Ischemic Cardiomyopathy.

    PubMed

    Shudo, Yasuhiro; Cohen, Jeffrey E; MacArthur, John W; Goldstone, Andrew B; Otsuru, Satoru; Trubelja, Alen; Patel, Jay; Edwards, Bryan B; Hung, George; Fairman, Alexander S; Brusalis, Christopher; Hiesinger, William; Atluri, Pavan; Hiraoka, Arudo; Miyagawa, Shigeru; Sawa, Yoshiki; Woo, Y Joseph

    2015-10-01

    There exists a substantial body of work describing cardiac support devices to mechanically support the left ventricle (LV); however, these devices lack biological effects. To remedy this, we implemented a cell sheet engineering approach utilizing chondrocytes, which in their natural environment produce a relatively elastic extracellular matrix (ECM) for a cushioning effect. Therefore, we hypothesized that a chondrocyte cell sheet applied to infarcted and borderzone myocardium will biologically enhance the ventricular ECM and increase elasticity to augment cardiac function in a model of ischemic cardiomyopathy (ICM). Primary articular cartilage chondrocytes of Wistar rats were isolated and cultured on temperature-responsive culture dishes to generate cell sheets. A rodent ICM model was created by ligating the left anterior descending coronary artery. Rats were divided into two groups: cell sheet transplantation (1.0 × 10(7) cells/dish) and no treatment. The cell sheet was placed onto the surface of the heart covering the infarct and borderzone areas. At 4 weeks following treatment, the decreased fibrotic extension and increased elastic microfiber networks in the infarct and borderzone areas correlated with this technology's potential to stimulate ECM formation. The enhanced ventricular elasticity was further confirmed by the axial stretch test, which revealed that the cell sheet tended to attenuate tensile modulus, a parameter of stiffness. This translated to increased wall thickness in the infarct area, decreased LV volume, wall stress, mass, and improvement of LV function. Thus, the chondrocyte cell sheet strengthens the ventricular biomechanical properties by inducing the formation of elastic microfiber networks in ICM, resulting in attenuated myocardial stiffness and improved myocardial function.

  9. A Tissue-Engineered Chondrocyte Cell Sheet Induces Extracellular Matrix Modification to Enhance Ventricular Biomechanics and Attenuate Myocardial Stiffness in Ischemic Cardiomyopathy

    PubMed Central

    Shudo, Yasuhiro; Cohen, Jeffrey E.; MacArthur, John W.; Goldstone, Andrew B.; Otsuru, Satoru; Trubelja, Alen; Patel, Jay; Edwards, Bryan B.; Hung, George; Fairman, Alexander S.; Brusalis, Christopher; Hiesinger, William; Atluri, Pavan; Hiraoka, Arudo; Miyagawa, Shigeru; Sawa, Yoshiki

    2015-01-01

    There exists a substantial body of work describing cardiac support devices to mechanically support the left ventricle (LV); however, these devices lack biological effects. To remedy this, we implemented a cell sheet engineering approach utilizing chondrocytes, which in their natural environment produce a relatively elastic extracellular matrix (ECM) for a cushioning effect. Therefore, we hypothesized that a chondrocyte cell sheet applied to infarcted and borderzone myocardium will biologically enhance the ventricular ECM and increase elasticity to augment cardiac function in a model of ischemic cardiomyopathy (ICM). Primary articular cartilage chondrocytes of Wistar rats were isolated and cultured on temperature-responsive culture dishes to generate cell sheets. A rodent ICM model was created by ligating the left anterior descending coronary artery. Rats were divided into two groups: cell sheet transplantation (1.0 × 107 cells/dish) and no treatment. The cell sheet was placed onto the surface of the heart covering the infarct and borderzone areas. At 4 weeks following treatment, the decreased fibrotic extension and increased elastic microfiber networks in the infarct and borderzone areas correlated with this technology's potential to stimulate ECM formation. The enhanced ventricular elasticity was further confirmed by the axial stretch test, which revealed that the cell sheet tended to attenuate tensile modulus, a parameter of stiffness. This translated to increased wall thickness in the infarct area, decreased LV volume, wall stress, mass, and improvement of LV function. Thus, the chondrocyte cell sheet strengthens the ventricular biomechanical properties by inducing the formation of elastic microfiber networks in ICM, resulting in attenuated myocardial stiffness and improved myocardial function. PMID:26154752

  10. Polarized ATP distribution in urothelial mucosal and serosal space is differentially regulated by stretch and ectonucleotidases.

    PubMed

    Yu, Weiqun

    2015-11-15

    Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch. PMID:26336160

  11. Piezo1 regulates mechanotransductive release of ATP from human RBCs.

    PubMed

    Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

    2015-09-22

    Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

  12. Piezo1 regulates mechanotransductive release of ATP from human RBCs

    PubMed Central

    Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E.; Wan, Jiandi

    2015-01-01

    Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca2+) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca2+ influx decrease significantly. Remarkably, a critical extracellular Ca2+ concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease. PMID:26351678

  13. Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis.

    PubMed

    Kwon, H J; Ohmiya, Y; Honma, K I; Honma, S; Nagai, T; Saito, K; Yasuda, K

    2012-03-08

    The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca(2+) oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion.

  14. Synchronized ATP oscillations have a critical role in prechondrogenic condensation during chondrogenesis

    PubMed Central

    Kwon, H J; Ohmiya, Y; Honma, K-i; Honma, S; Nagai, T; Saito, K; Yasuda, K

    2012-01-01

    The skeletal elements of embryonic limb are prefigured by prechondrogenic condensation in which secreted molecules such as adhesion molecules and extracellular matrix have crucial roles. However, how the secreted molecules are controlled to organize the condensation remains unclear. In this study, we examined metabolic regulation of secretion in prechondrogenic condensation, using bioluminescent monitoring systems. We here report on ATP oscillations in the early step of chondrogenesis. The ATP oscillations depended on both glycolysis and mitochondrial respiration, and their synchronization among cells were achieved via gap junctions. In addition, the ATP oscillations were driven by Ca2+ oscillations and led to oscillatory secretion in chondrogenesis. Blockade of the ATP oscillations prevented cellular condensation. Furthermore, the degree of cellular condensation increased with the frequency of ATP oscillations. We conclude that ATP oscillations have a critical role in prechondrogenic condensation by inducing oscillatory secretion. PMID:22402602

  15. Use of genetically encoded sensors to monitor cytosolic ATP/ADP ratio in living cells.

    PubMed

    Tarasov, Andrei I; Rutter, Guy A

    2014-01-01

    ATP is not only recognized as the universal energy "currency" in most cells but also plays a less well-known role as an intracellular and extracellular messenger. Here, we review novel approaches for measuring free ATP (or ATP/ADP ratios) in living mammalian cells by using genetically encoded sensors. We also discuss the key technical aspects of routine real-time ATP/ADP monitoring using as a model one of the last-generation fluorescent probes, a fusion protein commonly known as "Perceval." Finally, we present detailed guidelines for the simultaneous measurement of cytosolic ATP/ADP ratios and Ca(2+) concentrations alongside electrical parameters in individual pancreatic β cells, in which energy metabolism is tightly linked to plasma membrane excitability to control the secretion of insulin. With appropriate variations, this approach can be adapted to the study of cytosolic ATP/ADP ratios and Ca(2+) concentrations in malignant cells, two important aspects of oncometabolism.

  16. Myocardial Extracellular Volume Fraction with Dual-Energy Equilibrium Contrast-enhanced Cardiac CT in Nonischemic Cardiomyopathy: A Prospective Comparison with Cardiac MR Imaging.

    PubMed

    Lee, Hye-Jeong; Im, Dong Jin; Youn, Jong-Chan; Chang, Suyon; Suh, Young Joo; Hong, Yoo Jin; Kim, Young Jin; Hur, Jin; Choi, Byoung Wook

    2016-07-01

    Purpose To evaluate the feasibility of equilibrium contrast material-enhanced dual-energy cardiac computed tomography (CT) to determine extracellular volume fraction (ECV) in nonischemic cardiomyopathy (CMP) compared with magnetic resonance (MR) imaging. Materials and Methods This study was approved by the institutional review board; informed consent was obtained. Seven healthy subjects and 23 patients (six with hypertrophic CMP, nine with dilated CMP, four with amyloidosis, and four with sarcoidosis) (mean age ± standard deviation, 57.33 years ± 14.82; 19 male participants [63.3%]) were prospectively enrolled. Twelve minutes after contrast material injection (1.8 mL/kg at 3 mL/sec), dual-energy cardiac CT was performed. ECV was measured by two observers independently. Hematocrit levels were compared between healthy subjects and patients with the Mann-Whitney U test. In per-subject analysis, interobserver agreement for CT was assessed with the intraclass correlation coefficient (ICC), and intertest agreement between MR imaging and CT was assessed with Bland-Altman analysis. In per-segment analysis, Student t tests in the linear mixed model were used to compare ECV on CT images between healthy subjects and patients. Results Hematocrit level was 43.44% ± 1.80 for healthy subjects and 41.23% ± 5.61 for patients with MR imaging (P = .16) and 43.50% ± 1.92 for healthy subjects and 41.35% ± 5.92 for patients with CT (P = .15). For observer 1 in per-subject analysis, ECV was 34.18% ± 8.98 for MR imaging and 34.48% ± 8.97 for CT. For observer 2, myocardial ECV was 34.42% ± 9.03 for MR imaging and 33.98% ± 9.05 for CT. Interobserver agreement for ECV at CT was excellent (ICC = 0.987). Bland-Altman analysis between MR imaging and CT showed a small bias (-0.06%), with 95% limits of agreement of -1.19 and 1.79. Compared with healthy subjects, patients with hypertrophic CMP, dilated CMP, amyloidosis, and sarcoidosis had significantly higher myocardial ECV at dual

  17. Myocardial Extracellular Volume Fraction with Dual-Energy Equilibrium Contrast-enhanced Cardiac CT in Nonischemic Cardiomyopathy: A Prospective Comparison with Cardiac MR Imaging.

    PubMed

    Lee, Hye-Jeong; Im, Dong Jin; Youn, Jong-Chan; Chang, Suyon; Suh, Young Joo; Hong, Yoo Jin; Kim, Young Jin; Hur, Jin; Choi, Byoung Wook

    2016-07-01

    Purpose To evaluate the feasibility of equilibrium contrast material-enhanced dual-energy cardiac computed tomography (CT) to determine extracellular volume fraction (ECV) in nonischemic cardiomyopathy (CMP) compared with magnetic resonance (MR) imaging. Materials and Methods This study was approved by the institutional review board; informed consent was obtained. Seven healthy subjects and 23 patients (six with hypertrophic CMP, nine with dilated CMP, four with amyloidosis, and four with sarcoidosis) (mean age ± standard deviation, 57.33 years ± 14.82; 19 male participants [63.3%]) were prospectively enrolled. Twelve minutes after contrast material injection (1.8 mL/kg at 3 mL/sec), dual-energy cardiac CT was performed. ECV was measured by two observers independently. Hematocrit levels were compared between healthy subjects and patients with the Mann-Whitney U test. In per-subject analysis, interobserver agreement for CT was assessed with the intraclass correlation coefficient (ICC), and intertest agreement between MR imaging and CT was assessed with Bland-Altman analysis. In per-segment analysis, Student t tests in the linear mixed model were used to compare ECV on CT images between healthy subjects and patients. Results Hematocrit level was 43.44% ± 1.80 for healthy subjects and 41.23% ± 5.61 for patients with MR imaging (P = .16) and 43.50% ± 1.92 for healthy subjects and 41.35% ± 5.92 for patients with CT (P = .15). For observer 1 in per-subject analysis, ECV was 34.18% ± 8.98 for MR imaging and 34.48% ± 8.97 for CT. For observer 2, myocardial ECV was 34.42% ± 9.03 for MR imaging and 33.98% ± 9.05 for CT. Interobserver agreement for ECV at CT was excellent (ICC = 0.987). Bland-Altman analysis between MR imaging and CT showed a small bias (-0.06%), with 95% limits of agreement of -1.19 and 1.79. Compared with healthy subjects, patients with hypertrophic CMP, dilated CMP, amyloidosis, and sarcoidosis had significantly higher myocardial ECV at dual

  18. A titanium surface with nano-ordered spikes and pores enhances human dermal fibroblastic extracellular matrix production and integration of collagen fibers.

    PubMed

    Yamada, Masahiro; Kato, Eiji; Yamamoto, Akiko; Sakurai, Kaoru

    2016-02-02

    The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface. The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen. An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces. Well-organized, isotropically

  19. Carbon and energy metabolism of atp mutants of Escherichia coli.

    PubMed

    Jensen, P R; Michelsen, O

    1992-12-01

    The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis.

  20. Enhanced formation and impaired degradation of neutrophil extracellular traps in dermatomyositis and polymyositis: a potential contributor to interstitial lung disease complications

    PubMed Central

    Zhang, S; Shu, X; Tian, X; Chen, F; Lu, X; Wang, G

    2014-01-01

    Dermatomyositis (DM) and polymyosits (PM) are systemic autoimmune diseases whose pathogeneses remain unclear. Neutrophil extracellular traps (NETs) are reputed to play an important role in the pathogenesis of autoimmune diseases. This study tests the hypothesis that NETs may be pathogenic in DM/PM. Plasma samples from 97 DM/PM patients (72 DM, 25 PM) and 54 healthy controls were tested for the capacities to induce and degrade NETs. Plasma DNase I activity was tested to further explore possible reasons for the incomplete degradation of NETs. Results from 35 DM patients and seven PM patients with interstitial lung disease (ILD) were compared with results from DM/PM patients without ILD. Compared with control subjects, DM/PM patients exhibited a significantly enhanced capacity for inducing NETs, which was supported by elevated levels of plasma LL-37 and circulating cell-free DNA (cfDNA) in DM/PM. NETs degradation and DNase I activity were also decreased significantly in DM/PM patients and were correlated positively. Moreover, DM/PM patients with ILD exhibited the lowest NETs degradation in vitro due to the decrease in DNase I activity. DNase I activity in patients with anti-Jo-1 antibodies was significantly lower than in patients without. Glucocorticoid therapy seems to improve DNase I activity. Our findings demonstrate that excessively formed NETs cannot be degraded completely because of decreased DNase I activity in DM/PM patients, especially in patients with ILD, suggesting that abnormal regulation of NETs may be involved in the pathogenesis of DM/PM and could be one of the factors that initiate and aggravate ILD. PMID:24611519

  1. 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

  2. Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions

    PubMed Central

    Kean, Thomas J.; Dennis, James E.

    2015-01-01

    Background Current tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential. Methods Porcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically. Results Human chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions. Conclusions

  3. Adenosine and ATP Link PCO2 to Cortical Excitability via pH

    PubMed Central

    Dulla, Chris G.; Dobelis, Peter; Pearson, Tim; Frenguelli, Bruno G.; Staley, Kevin J.; Masino, Susan A.

    2007-01-01

    Summary In addition to affecting respiration and vascular tone, deviations from normal CO2 alter pH, consciousness, and seizure propensity. Outside the brainstem, however, the mechanisms by which CO2 levels modify neuronal function are unknown. In the hippocampal slice preparation, increasing CO2, and thus decreasing pH, increased the extracellular concentration of the endogenous neuromodulator adenosine and inhibited excitatory synaptic transmission. These effects involve adenosine A1 and ATP receptors and depend on decreased extracellular pH. In contrast, decreasing CO2 levels reduced extracellular adenosine concentration and increased neuronal excitability via adenosine A1 receptors, ATP receptors, and ecto-ATPase. Based on these studies, we propose that CO2-induced changes in neuronal function arise from a pH-dependent modulation of adenosine and ATP levels. These findings demonstrate a mechanism for the bidirectional effects of CO2 on neuronal excitability in the forebrain. PMID:16364904

  4. 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

  5. Extracellular Ca2+ Acts as a Mediator of Communication from Neurons to Glia

    PubMed Central

    Torres, Arnulfo; Wang, Fushun; Xu, Qiwu; Fujita, Takumi; Dobrowolski, Radoslaw; Willecke, Klaus; Takano, Takahiro; Nedergaard, Maiken

    2013-01-01

    Defining the pathways through which neurons and astrocytes communicate may contribute to the elucidation of higher central nervous system functions. We investigated the possibility that decreases in extracellular calcium ion concentration ([Ca2+]e) that occur during synaptic transmission might mediate signaling from neurons to glia. Using noninvasive photolysis of the photolabile Ca2+ buffer diazo-2 {N-[2-[2-[2-[bis(carboxymethyl)amino]-5-(diazoacetyl)phenoxy]ethoxy]-4-methylphenyl]-N-(carboxymethyl)-, tetrapotassium salt} to reduce [Ca2+]e or caged glutamate to simulate glutamatergic transmission, we found that a local decline in extracellular Ca2+ triggered astrocytic adenosine triphosphate (ATP) release and astrocytic Ca2+ signaling. In turn, activation of purinergic P2Y1 receptors on a subset of inhibitory interneurons initiated the generation of action potentials by these interneurons, thereby enhancing synaptic inhibition. Thus, astrocytic ATP release evoked by an activity-associated decrease in [Ca2+]e may provide a negative feedback mechanism that potentiates inhibitory transmission in response to local hyperexcitability. PMID:22275221

  6. Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.

    PubMed

    Skals, Marianne; Bjaelde, Randi G; Reinholdt, Jesper; Poulsen, Knud; Vad, Brian S; Otzen, Daniel E; Leipziger, Jens; Praetorius, Helle A

    2014-07-01

    ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

  7. 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.

  8. Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea

    PubMed Central

    Uzlaner, Natalya; Priel, Zvi

    1999-01-01

    Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation.Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin.The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3′, 5′-cyclic monophosphothioate triethylamine (Rp-cAMPS).Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i.Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin.The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP. PMID:10066932

  9. The C421A (Q141K) polymorphism enhances the 3'-untranslated region (3'-UTR)-dependent regulation of ATP-binding cassette transporter ABCG2.

    PubMed

    Ripperger, Anne; Benndorf, Ralf A

    2016-03-15

    The impact of the gout-causing C421A (Q141K) single nucleotide polymorphism (SNP) on ABC transporter ABCG2 expression and function has been extensively characterized. However, the influence of the C421A SNP on 3'-UTR-dependent ABCG2 regulation has not been analysed so far. To elucidate this matter, we generated vectors for expression of either the ABCG2 coding sequence (ORF) or the ABCG2 ORF fused to its 3'-UTR, inserted the C421A mutation via site-directed mutagenesis and expressed wild-type and C421A-mutated ABCG2 transcripts in HEK293-Tet-On cells. As shown previously, the C421A SNP significantly reduced ABCG2 protein levels in ABCG2 ORF-transfected HEK293-Tet-On cells. Interestingly, the presence of the 3'-UTR in the ABCG2 transcript dramatically reduced ABCG2 protein content in cells transfected with the C421A variant but not significantly in those transfected with ABCG2 wild-type sequence, whereas ABCG2 mRNA levels were similar. siRNA-mediated DICER1 knockdown to reduce cellular microRNA biogenesis and selective mutation of putative microRNA binding sites within the ABCG2 3'-UTR partially antagonized C421A-associated reduction of ABCG2 protein content but did not significantly affect wild-type ABCG2 protein levels. In addition, antagomir-mediated inhibition of two microRNAs (hsa-miR-519c and hsa-miR-328) again partially reversed C421A-associated ABCG2 translational repression, thereby indicating that the C421A SNP may facilitate microRNA-dependent repression of ABCG2 protein translation. We conclude from our results that the C421A SNP may lead to reduced ABCG2 protein levels not only by affecting cellular protein stability but also via enhanced microRNA-dependent ABCG2 repression. Moreover, tissue-specific variation in ABCG2 3'-UTR processing may profoundly affect ABCG2 expression levels in individuals carrying the C421A mutation. PMID:26903388

  10. Interdependence of ATP signalling and pannexin channels; the servant was really the master all along?

    PubMed

    Jackson, Michael F

    2015-12-15

    Pannexin channels are recognized as important conduits for the release of ATP, which contributes to purinergic signalling. Pathologically, ATP release via these channels acts as a find-me signal for apoptotic cell clearance. Accordingly, there is considerable and growing interest in understanding the function and regulation of pannexin channels. In a recent issue of the Biochemical Journal, Boyce et al. provide evidence that the surface expression of pannexin channels is regulated by extracellular ATP. They propose a model in which ATP triggers pannexin channel internalization through a pathway involving clathrin- and caveolin-independent entry into early endosomes. Intriguingly, their evidence suggests that internalization is initiated through the association of ATP with pannexin channels themselves as well as ionotropic purinergic receptor 7 (P2X7) receptors. PMID:26613946

  11. Imaging of ATP membrane transport with dual micro-disk electrodes and scanning electrochemical microscopy.

    PubMed

    Kueng, Angelika; Kranz, Christine; Mizaikoff, Boris

    2005-08-15

    Extracellular adenosine-5'-triphosphate (ATP) is involved in a variety of relevant regulatory mechanisms at a cellular level and has therefore been focus of extensive research. One of the major challenges associated with measuring this key regulatory analyte is the ability to detect and localize extracellular ATP with sufficient spatial and temporal resolution in physiological environments. In this study, scanning electrochemical microscopy (SECM) utilizing an amperometric micro-biosensor based on co-immobilization of the enzymes glucose oxidase and hexokinase is applied for imaging ATP transport through a porous polycarbonate membrane under physiologically relevant conditions. The enzymatic biosensor operates on competitive consumption of the substrate glucose between the immobilized enzymes glucose oxidase and hexokinase involving ATP as a co-substrate. Quantitative determination of the ATP concentration is based on a linear correlation between the glucose consumption and the ATP level. Integration of the amperometric ATP micro-biosensor into a dual micro-disk electrode configuration is achieved by immobilizing the enzymes at one of the micro-disk electrodes while the second disk serves as an unmodified amperometric probe for controlled positioning of the micro-biosensor in close proximity to the sample surface enabling quantification of the obtained current signal. PMID:16023962

  12. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  13. Sources of Intravascular ATP During Exercise in Humans: Critical Role for Skeletal Muscle Perfusion

    PubMed Central

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

    2013-01-01

    Exercise hyperemia is regulated by several factors and one factor known to increase with exercise that evokes powerful vasomotor action is extracellular ATP. The origination of ATP detectable in plasma from exercising muscle of humans is, however, a matter of debate and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells, and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise further increases ATP vs exercise alone. We show that increased SNA via −40mmHg lower body negative pressure (LBNP) at rest does not increase plasma ATP (51±8 vs 58±7 nmol/L with LBNP), nor does it increase [ATP] above levels observed during rhythmic handgrip exercise (79±11 exercise alone vs 71±8 nmol/L with LBNP). Secondly, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We identify that complete obstruction of blood flow to contracting muscle abolishes exercise-mediated increases in plasma ATP (90±19 to 49±12 nmol/L), and further, that cessation of blood flow prior to exercise completely inhibits the typical rise in ATP (3 vs 61%; obstructed vs intact perfusion). The lack of ATP change during occlusion occurred in the face of continued muscle work and elevated SNA, indicating the rise of intravascular ATP is not resultant from these extravascular sources. Our collective observations indicate that the elevation in extracellular ATP observed in blood during exercise is unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather is dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential and points toward an important role for blood sources (e.g. red blood cells) in augmenting and

  14. Neutrophils as sources of extracellular nucleotides: Functional consequences at the vascular interface

    PubMed Central

    Eltzschig, Holger K.; MacManus, Christopher F.; Colgan, Sean P.

    2009-01-01

    Nucleotide signaling is currently an area of intense investigation. Extracellular ATP liberated during hypoxia or inflammation can either signal directly to purinergic receptors or, following phosphohydrolytic metabolism, can activate surface adenosine (Ado) receptors. Given the association of polymorphonuclear leukocytes (PMN) with adenine nucleotide / nucleoside signaling in the inflammatory milieu, it was recently demonstrated that PMN actively release ATP via a connexin 43 (Cx43) hemichannel-dependent mechanism. Here we review the mechanisms of ATP release and subsequent functional implications of ATP metabolism at the interface between PMN and vascular endothelial cells during inflammation and in hypoxia. PMID:18436149

  15. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    PubMed

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose. PMID:27250218

  16. Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins.

    PubMed

    Vidak, Sandra; Kubben, Nard; Dechat, Thomas; Foisner, Roland

    2015-10-01

    Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin.

  17. Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins.

    PubMed

    Vidak, Sandra; Kubben, Nard; Dechat, Thomas; Foisner, Roland

    2015-10-01

    Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin. PMID:26443848

  18. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil γ-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the γ shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  19. Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKKβ-AMPK-SIRT1 Signaling Pathway

    PubMed Central

    Zhu, Yuanfeng; Yang, Yongjun; Chen, Xiaoli; Fan, Shijun; Chen, Qian; Zheng, Jiang

    2016-01-01

    Activated macrophages are the primary sources of IL-12, a key cytokine bridging innate and adaptive immunity. However, macrophages produce low amounts of IL-12 upon stimulation and the underlying regulatory mechanism remains unclear. In this study, we found a new calcium-dependent mechanism that controlled IL-12 production in LPS-treated murine macrophages. First, LPS was demonstrated to induce extracellular calcium entry in murine peritoneal macrophages and inhibition of calcium influx resulted in marked enhancement in IL-12 production. Then, withdrawal of extracellular calcium was found to suppress CaMKKβ and AMPK activation triggered by LPS while chemical inhibition or genetic knockdown of these two kinases augmented LPS induced IL-12 production. AMPK activation increased the NAD+/NADH ratio and activated Sirtuin 1 (SIRT1), a NAD+-dependent deacetylating enzyme and negative regulator of inflammation. Chemical inhibitor or siRNA of SIRT1 enhanced IL-12 release while its agonist suppressed IL-12 production. Finally, it was found that SIRT1 selectively affected the transcriptional activity of NF-κB which thereby inhibited IL-12 production. Overall, our study demonstrates a new role of transmembrane calcium mobilization in immunity modulation such that inhibition of calcium influx leads to impaired activation of CaMKKβ-AMPK-SIRT1 signaling pathway which lifts restriction on NF-κB activation and results in enhanced IL-12 production. PMID:27313401

  20. Hemolysis is a primary ATP-release mechanism in human erythrocytes.

    PubMed

    Sikora, Jacek; Orlov, Sergei N; Furuya, Kishio; Grygorczyk, Ryszard

    2014-09-25

    The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

  1. Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

    PubMed

    Nomura, Johji; So, Alexander; Tamura, Mizuho; Busso, Nathalie

    2015-12-15

    Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. PMID:26546608

  2. ATP as a biomarker of viable microorganisms in clean-room facilities

    NASA Technical Reports Server (NTRS)

    Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T.; Kern, Roger

    2003-01-01

    A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.

  3. Nonmetal haptens induce ATP release from keratinocytes through opening of pannexin hemichannels by reactive oxygen species.

    PubMed

    Onami, Kaoru; Kimura, Yutaka; Ito, Yumiko; Yamauchi, Takeshi; Yamasaki, Kenshi; Aiba, Setsuya

    2014-07-01

    Although extracellular adenosine 5'-triphosphate (eATP) has a crucial role in the sensitization phase of contact hypersensitivity (CHS), the mechanism by which hapten causes keratinocyte cell death and ATP release is unknown. We examined the time course of cell death, reactive oxygen species (ROS) production, and ATP release in HaCaT cells and in normal human keratinocytes after exposure to nonmetal haptens, NiCl2, or irritants. Both haptens and irritants caused cell death of keratinocytes but with different time courses. N-acetylcysteine (NAC) significantly reduced only nonmetal hapten-induced cell death as assessed by propidium iodide exclusion. We examined the effects of antioxidants and pannexin (Panx) inhibitors on cell death, ROS production, and ATP release by chemical-treated HaCaT cells. Nonmetal hapten-induced cell death, but not NiCl2- or irritant-related cell death, was dependent on reactivity to thiol residues in the cells. NAC reduced cell death and ATP release, whereas antioxidants and Panx inhibitors did not inhibit cell death but significantly attenuated ATP release. Panx1 small interfering RNA (siRNA) also suppressed ATP release from hapten-exposed HaCaT cells. Intraperitoneal injection of a Panx1 inhibitor attenuated murine CHS. These findings suggest that nonmetal hapten reactivity to thiol residues causes membrane disruption of keratinocytes and ROS production that leads to ATP release through opening of Panx hemichannels. PMID:24531690

  4. Defective roles of ATP7B missense mutations in cellular copper tolerance and copper excretion.

    PubMed

    Zhu, Min; Dong, Yi; Ni, Wang; Wu, Zhi-Ying

    2015-07-01

    Wilson's disease (WD) is a hereditary disorder of copper metabolism resulting from mutations within ATP7B. Clinical investigations showed that ATP7B missense mutations cause a wide variety of symptoms in WD patients, which implies that those mutations might affect ATP7B function in a number of ways and each would have deleterious consequences on normal copper distribution and lead to WD. Nonetheless, it is still unknown about the influences of those mutations on ATP7B function of increasing copper excretion and enhancing cellular copper tolerance. Here we established the stable expression cell lines of wild-type (WT) ATP7B and its four missense mutants (R778L, R919G, T935M and P992L), tested cellular copper tolerance and copper excretion using those cell lines, and also observed cellular distribution of WT ATP7B proteins and those mutants in transiently transfected cells. We found that extrinsic expressing WT ATP7B reduced CuCl2-induced copper accumulation and enhanced cellular copper tolerance by accelerating copper excretion, which was selectively compromised by R778L and P992L mutations. Further investigation showed that R778L mutation disrupted the subcellular localization and trafficking of ATP7B proteins, whereas P992L mutation only affected the trafficking of ATP7B. This indicates that ATP7B missense mutants have distinct effects on cellular copper tolerance.

  5. Activation by ATP of a P2U 'nucleotide' receptor in an exocrine cell.

    PubMed Central

    Martin, S. C.; Shuttleworth, T. J.

    1995-01-01

    1. We employed the perforated patch whole-cell technique to investigate the effects of ATP and other related nucleotides on membrane conductances in avian exocrine salt gland cells. 2. ATP (10 microM-1 mM) evoked an increase in maxi-K+ and Cl- conductances with a reversal potential of -35 mV. At lower concentrations of ATP (< or = 100 microM) responses were generally oscillatory with a sustained response observed at higher concentrations (> or = 200 microM). 3. Both oscillatory and sustained responses were abolished by the removal of bath Ca2+. In cells preincubated in extracellular saline containing reduced Ca2+, the application of ATP resulted in a transient increase in current. 4. As increasing concentrations of ATP (and related nucleotides) evoked a graded sequence of events with little run-down we were able to establish a rank order of potency in single cells. The order of potency of ATP analogues and agonists of the various P2-receptor subtypes was UTP > ATP = 2-methylthio-ATP > ADP. Adenosine (1 microM-1 mM), AMP (1 microM-1 mM), alpha,beta-methylene-ATP (1 microM-1 mM) and beta,gamma-methylene-ATP (1 microM-1 mM) were without effect. 5. In conclusion, although unable to preclude a role for a P2Y-receptor, our results suggest that ATP binds to a P2U-receptor increasing [Ca2+]i and subsequently activating Ca(2+)-sensitive K+ and Cl- currents. PMID:7670734

  6. ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action.

    PubMed

    Betzenhauser, Matthew J; Wagner, Larry E; Iwai, Miwako; Michikawa, Takayuki; Mikoshiba, Katsuhiko; Yule, David I

    2008-08-01

    ATP enhances Ca(2+) release from inositol (1,4,5)-trisphosphate receptors (InsP(3)R). However, the three isoforms of InsP(3)R are reported to respond to ATP with differing sensitivities. Ca(2+) release through InsP(3)R1 is positively regulated at lower ATP concentrations than InsP(3)R3, and InsP(3)R2 has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP(3)R2 and InsP(3)R3 expressed in isolation on a null background in DT40 InsP(3)R knockout cells. We report that the Ca(2+)-releasing activity as well as the single channel open probability of InsP(3)R2 was enhanced by ATP, but only at submaximal InsP(3) levels. Further, InsP(3)R2 was more sensitive to ATP modulation than InsP(3)R3 under similar experimental conditions. Mutations in the ATPB sites of InsP(3)R2 and InsP(3)R3 were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP(3)R3 had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP(3)R2 eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP(3)R2.

  7. Does ATP cross the cell plasma membrane.

    PubMed Central

    Chaudry, I. H.

    1982-01-01

    Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes. PMID:7051582

  8. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

    PubMed

    Kasuya, Go; Fujiwara, Yuichiro; Takemoto, Mizuki; Dohmae, Naoshi; Nakada-Nakura, Yoshiko; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-02-01

    P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors. PMID:26804916

  9. An extracellular polymer at the interface of magnetic bioseparations.

    PubMed

    Dhadge, Vijaykumar L; Morgado, Patricia I; Freitas, Filomena; Reis, Maria A; Azevedo, Ana; Aires-Barros, Raquel; Roque, A Cecilia A

    2014-11-01

    FucoPol, a fucose-containing extracellular polysaccharide (EPS) produced by bacterium Enterobacter A47 using glycerol as the carbon source, was employed as a coating material for magnetic particles (MPs), which were subsequently functionalized with an artificial ligand for the capture of antibodies. The performance of the modified MPs (MP-EPS-22/8) for antibody purification was investigated using direct magnetic separation alone or combined with an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran. In direct magnetic capturing, and using pure protein solutions of human immunoglobulin G (hIgG) and bovine serum albumin (BSA), MP-EPS-22/8 bound 120 mg hIgG g(-1) MPs, whereas with BSA only 10 ± 2 mg BSA g(-1) MPs was achieved. The hybrid process combining both the ATPS and magnetic capturing leads to a good performance for partitioning of hIgG in the desired phase as well as recovery by the magnetic separator. The MPs were able to bind 145 mg of hIgG g(-1) of particles which is quite high when compared with direct magnetic separation. The theoretical maximum capacity was calculated to be 410 ± 15 mg hIgG adsorbed g(-1) MPs with a binding affinity constant of 4.3 × 10(4) M(-1). In multiple extraction steps, the MPs bound 92% of loaded hIgG with a final purity level of 98.5%. The MPs could easily be regenerated, recycled and re-used for five cycles with only minor loss of capacity. FucoPol coating allowed both electrostatic and hydrophobic interactions with the antibody contributing to enhance the specificity for the targeted products. PMID:25185582

  10. An extracellular polymer at the interface of magnetic bioseparations.

    PubMed

    Dhadge, Vijaykumar L; Morgado, Patricia I; Freitas, Filomena; Reis, Maria A; Azevedo, Ana; Aires-Barros, Raquel; Roque, A Cecilia A

    2014-11-01

    FucoPol, a fucose-containing extracellular polysaccharide (EPS) produced by bacterium Enterobacter A47 using glycerol as the carbon source, was employed as a coating material for magnetic particles (MPs), which were subsequently functionalized with an artificial ligand for the capture of antibodies. The performance of the modified MPs (MP-EPS-22/8) for antibody purification was investigated using direct magnetic separation alone or combined with an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran. In direct magnetic capturing, and using pure protein solutions of human immunoglobulin G (hIgG) and bovine serum albumin (BSA), MP-EPS-22/8 bound 120 mg hIgG g(-1) MPs, whereas with BSA only 10 ± 2 mg BSA g(-1) MPs was achieved. The hybrid process combining both the ATPS and magnetic capturing leads to a good performance for partitioning of hIgG in the desired phase as well as recovery by the magnetic separator. The MPs were able to bind 145 mg of hIgG g(-1) of particles which is quite high when compared with direct magnetic separation. The theoretical maximum capacity was calculated to be 410 ± 15 mg hIgG adsorbed g(-1) MPs with a binding affinity constant of 4.3 × 10(4) M(-1). In multiple extraction steps, the MPs bound 92% of loaded hIgG with a final purity level of 98.5%. The MPs could easily be regenerated, recycled and re-used for five cycles with only minor loss of capacity. FucoPol coating allowed both electrostatic and hydrophobic interactions with the antibody contributing to enhance the specificity for the targeted products.

  11. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    PubMed

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process. PMID:27287557

  12. Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration.

    PubMed

    Taurino, Federica; Giannoccaro, Caterina; Sardanelli, Anna Maria; Cavallo, Alessandro; De Luca, Elisa; Santacroce, Salvatore; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2016-08-15

    ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming phase of liver regeneration, 3 h after partial hepatectomy, liver mitochondria showed a marked lowering of the ATP synthase protein level that was reflected in the impairment of both ATP synthesis and hydrolysis. The ecto-ATP synthase level, in 3 h partial hepatectomized hepatocytes, was decreased similarly to the level of the mitochondrial ATP synthase, associated with a lowering of the ecto-ATP hydrolase activity coupled to proton influx. Noteworthily, the ecto-ATP synthase activity coupled to proton efflux was completely inhibited in 3 h partial hepatectomized hepatocytes, even in the presence of a marked intracellular acidification that would sustain it as in control and sham-operated hepatocytes. At the end of the liver regeneration, 7 days after partial hepatectomy, the level and the catalytic activities of the ecto- and mitochondrial ATP synthase reached the control and sham-operated values. The specific modulation of hepatocyte ecto-ATP synthase catalytic activities during liver regeneration priming phase may modulate the extracellular ADP/ATP levels and/or proton influx/efflux trafficking, making hepatocyte ecto-ATP synthase a candidate for a novel player in the liver regeneration process.

  13. Adenosine enhances sweet taste through A2B receptors in the taste bud

    PubMed Central

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

  14. Adenosine enhances sweet taste through A2B receptors in the taste bud.

    PubMed

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

  15. Two non-vesicular ATP release pathways in the mouse erythrocyte membrane

    PubMed Central

    Qiu, Feng; Wang, Junjie; Spray, David C.; Scemes, Eliana; Dahl, Gerhard

    2011-01-01

    Erythrocytes are exceptionally suited for analysis of non-exocytotic release mechanisms of ATP, because these cells under physiological conditions lack vesicles. Previous studies have indicated, that Pannexin1 (Panx1) provides a key ATP permeation pathway in many cell types, including human and frog erythrocytes. Here we show that erythrocytes of Panx1-/- mice lend further support to this conclusion. However, ATP release, although attenuated, was still observed in Panx1-/- mouse erythrocytes. In contrast to Panx1+/+ cells, this release was not correlated with uptake of extracellularly applied dyes, was insensitive to Panx1 channel blockers, and was inhibited by dipyridamole and stimulated by iloprost. Thus, in erythrocytes, two independent pathways mediate the release of ATP. We also show that glyburide is a strong inhibitor of Panx1 channels. PMID:21983290

  16. Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

    PubMed Central

    Maniam, S; Coutts, A S; Stratford, M R; McGouran, J; Kessler, B; La Thangue, N B

    2015-01-01

    Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration. PMID:25168243

  17. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    SciTech Connect

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-05-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes /sup 14/C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg/sup 2 +/. Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-..gamma..-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated.

  18. Application of ATP measurements to the microbiological evaluation of a petroleum reservoir

    SciTech Connect

    Jones, P.M.

    1981-06-01

    The objective of the work reported in this document was to determine whether the bioluminescent luciferin/luciferase based adenosine triphosphate (ATP) assay could be used as a rapid field tests for determining the presence and numbers of microorganisms in oil field fluids. The ATP-photometric technique employed is based on the ATP-mediated bioluminescent oxidation of firefly luciferin. Light production is stoichiometrically related to ATP concentration; ATP concentration is related to numbers of living organisms present in a sample. Samples used in this study comprised reservoir fluids collected from several Southern California oilfields. Based on experimental evidence, it was concluded that the ATP assay could be profitably applied to Microbially Enhanced Oil Recovery (MEOR) process monitoring and control. The theoretical basis for the assay, field-usage methodologies, and fundamentals of data interpretation are presented to make the document usable as a field manual.

  19. 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. PMID:26986520

  20. 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.

  1. Impaired skeletal muscle blood flow control with advancing age in humans: attenuated ATP release and local vasodilation during erythrocyte deoxygenation

    PubMed Central

    Kirby, Brett S.; Crecelius, Anne R.; Voyles, Wyatt F.; Dinenno, Frank A.

    2012-01-01

    Rationale Skeletal muscle blood flow is coupled with the oxygenation state of hemoglobin in young adults, whereby the erythrocyte functions as an oxygen sensor and releases ATP during deoxygenation to evoke vasodilation. Whether this function is impaired in humans of advanced age is unknown. Objective To test the hypothesis that older adults demonstrate impaired muscle blood flow and lower intravascular ATP during conditions of erythrocyte deoxygenation. Methods and Results We show impaired forearm blood flow (FBF) responses during two conditions of erythrocyte deoxygenation (systemic hypoxia and graded handgrip exercise) with age, and this is due to reduced local vasodilation. In young adults, both hypoxia and exercise significantly increased venous [ATP] and ATP effluent (FBF × [ATP]) draining skeletal muscle. In contrast, hypoxia and exercise did not increase [ATP]v in older adults, and both [ATP]v and ATP effluent were substantially reduced compared with young despite similar levels of deoxygenation. Next, we demonstrate that this cannot be explained by augmented extracellular ATP hydrolysis in whole blood with age. Finally, we found that deoxygenation-mediated ATP release from isolated erythrocytes is essentially non-existent in older adults. Conclusions Skeletal muscle blood flow during conditions of erythrocyte deoxygenation is markedly reduced in aging humans, and reductions in plasma ATP and erythrocyte-mediated ATP release may be a novel mechanism underlying impaired vasodilation and oxygen delivery during hypoxemia with advancing age. Because aging is associated with elevated risk of ischemic cardiovascular disease and exercise intolerance, interventions targeting erythrocyte-mediated ATP release may offer therapeutic potential. PMID:22647875

  2. Mechanism of action of ATP on canine pulmonary vagal C fibre nerve terminals.

    PubMed Central

    Pelleg, A; Hurt, C M

    1996-01-01

    1. The effects of extracellular adenosine 5'-triphosphate (ATP) on pulmonary vagal afferent fibres (n = 46) was studied in a canine model in vivo (n = 38). 2. ATP (3-6 mumol kg-1), administered as a rapid bolus into the right atrium, elicited a transient burst of action potentials in cervical vagal fibres, which was not affected by either blockade of ganglionic transmission (hexamethonium) or a drop in arterial blood pressure (nitroglycerine). 3. The fibres with ATP-sensitive terminals were otherwise quiescent with no activity related to either cardiac or respiratory cycles and their conduction velocity was 0.85 +/- 0.13 m s-1 (n = 7). 4. Inflation of the lungs to 2-3 times the tidal volume triggered brief bursts of action potentials in these fibres. 5. Capsaicin (10 micrograms kg-1), given as a rapid bolus into the right atrium, elicited a burst of action potentials in these ATP-sensitive fibres. 6. Smaller amounts of ATP and capsaicin (0.5-3 mumol kg-1 and 1-5 micrograms kg-1, respectively) had similar effects when the two compounds were given into the right pulmonary artery. 7. Adenosine, adenosine 5'-monophosphate, or adenosine 5'-diphosphate did not excite these fibres (n = 30). 8. The non-degradable analogue of ATP alpha,beta-methylene ATP (alpha,beta-mATP) was tenfold more potent than ATP while beta,gamma-methylene ATP (beta,gamma-mATP) was in active. 9. The selective P2x-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid markedly attenuated the effect of ATP but not of capsaicin. The P2Y-purinoceptor antagonist Reactive Blue 2 was without effect. 10. Pretreatment with pertussis toxin (PTX) did not affect this action of ATP. 11. In the canine lungs ATP activates vagal C fibre nerve terminals. This action is mediated by P2X-purinoceptors and is independent of a PTX-sensitive guanine nucleotide binding protein (G protein). PMID:8745294

  3. The modulation of extracellular superoxide dismutase in the specifically enhanced cellular immune response against secondary challenge of Vibrio splendidus in Pacific oyster (Crassostrea gigas).

    PubMed

    Liu, Conghui; Zhang, Tao; Wang, Lingling; Wang, Mengqiang; Wang, Weilin; Jia, Zhihao; Jiang, Shuai; Song, Linsheng

    2016-10-01

    Extracellular superoxide dismutase (EcSOD) is a copper-containing glycoprotein playing an important role in antioxidant defense of living cells exposed to oxidative stress, and also participating in microorganism internalization and cell adhesion in invertebrates. EcSOD from oyster (designated CgEcSOD) had been previously reported to bind lipopolysaccharides (LPS) and act as a bridge molecule in Vibrio splendidus internalization. Its mRNA expression pattern, PAMP binding spectrum and microorganism binding capability were examined in the present study. The mRNA expression of CgEcSOD in hemocytes was significantly up-regulated at the initial phase and decreased sharply at 48 h post V. splendidus stimulation. The recombinant CgEcSOD protein (rCgEcSOD) could bind LPS, PGN and poly (I:C), as well as various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica at the presence of divalent metal ions Cu(2+). After the secondary V. splendidus stimulation, the mRNA and protein of CgEcSOD were both down-regulated significantly. The results collectively indicated that CgEcSOD could not only function in the immune recognition, but also might contribute to the immune priming of oyster by inhibiting the foreign microbe invasion through a specific down-regulation. PMID:27268574

  4. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    PubMed

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

  5. Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

    PubMed Central

    Cohen, Matthew R.; Johnson, William M.; Pilat, Jennifer M.; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E.

    2015-01-01

    Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca2+-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca2+-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca2+ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca2+ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

  6. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

    PubMed Central

    Dash, Biplab Kumar; Rahman, M. Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time. PMID:26180814

  7. Artificial extracellular matrices of collagen and sulphated hyaluronan enhance the differentiation of human mesenchymal stem cells in the presence of dexamethasone.

    PubMed

    Hintze, V; Miron, A; Möller, S; Schnabelrauch, M; Heinemann, S; Worch, H; Scharnweber, D

    2014-04-01

    In this study we investigated the potential of artificial extracellular matrix (aECM) coatings containing collagen II and two types of glycosaminoglycan (GAGs) with different degrees of sulphation to promote human bone formation in biomedical applications. To this end their impact on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assessed. The cell proliferation was found to be significantly retarded in the first 14 days of culture on surfaces coated with collagen II and GAGs (coll-II/GAG) as compared to tissue culture polystyrol (TCPS) and those coated with collagen II. At later time points it only tended to be retarded on coll-II/sHya3.1. Heat-inactivation of the serum significantly reduced cell numbers on collagen II and coll-II/sHya3.1. Alkaline phosphatase (ALP) activity and calcium deposition, on the other hand, were higher for coatings containing sHya3.1 and were not significantly changed by heat-inactivation of the serum. Expression levels of the bone matrix proteins bone sialoprotein (BSP-II) and osteopontin (OP) were also increased on aECM coatings as compared to TCPS, which further validated the differentiation of hMSCs towards the osteogenic lineage. These observations reveal that aECM coatings, in particular those containing sHya3.1, are suitable to promote the osteogenic differentiation of hMSCs.

  8. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    PubMed

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  9. 2´,3´-Dialdehyde of ATP, ADP, and adenosine inhibit HIV-1 reverse transcriptase and HIV-1 replication.

    PubMed

    Schachter, Julieta; Valadao, Ana Luiza Chaves; Aguiar, Renato Santana; Barreto-de-Souza, Victor; Rossi, Atila Duque; Arantes, Pablo Ricardo; Verli, Hugo; Quintana, Paula Gabriela; Heise, Norton; Tanuri, Amilcar; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis

    2014-01-01

    The 2´3´-dialdehyde of ATP or oxidized ATP (oATP) is a compound known for specifically making covalent bonds with the nucleotide-binding site of several ATP-binding enzymes and receptors. We investigated the effects of oATP and other oxidized purines on HIV-1 infection and we found that this compound inhibits HIV-1 and SIV infection by blocking early steps of virus replication. oATP, oxidized ADP (oADP), and oxidized Adenosine (oADO) impact the natural activity of endogenous reverse transcriptase enzyme (RT) in cell free virus particles and are able to inhibit viral replication in different cell types when added to the cell cultures either before or after infection. We used UFLC-UV to show that both oADO and oATP can be detected in the cell after being added in the extracellular medium. oATP also suppresses RT activity and replication of the HIV-1 resistant variants M184V and T215Y. We conclude that oATP, oADP and oADO display anti HIV-1 activity that is at in least in part due to inhibitory activity on HIV-1 RT.

  10. Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes.

    PubMed

    Bowser, David N; Khakh, Baljit S

    2007-06-01

    Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. Here we explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. We find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between approximately 100 and 250 microm, and approximately 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Our data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

  11. Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

    PubMed

    Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

    2014-03-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories.

  12. Pathophysiological Role of Extracellular Purinergic Mediators in the Control of Intestinal Inflammation

    PubMed Central

    Kurashima, Yosuke; Kiyono, Hiroshi

    2015-01-01

    Purinergic mediators such as adenosine 5′-triphosphate (ATP) are released into the extracellular compartment from damaged tissues and activated immune cells. They are then recognized by multiple purinergic P2X and P2Y receptors. Release and recognition of extracellular ATP are associated with both the development and the resolution of inflammation and infection. Accumulating evidence has recently suggested the potential of purinergic receptors as novel targets for drugs for treating intestinal disorders, including intestinal inflammation and irritable bowel syndrome. In this review, we highlight recent findings regarding the pathophysiological role of purinergic mediators in the development of intestinal inflammation. PMID:25944982

  13. Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

    PubMed Central

    Suidan, H S; Murrell, R D; Tolkovsky, A M

    1991-01-01

    The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered. PMID:1848792

  14. Antiphospholipid Antibodies Bind ATP: A putative Mechanism for the Pathogenesis of Neuronal Dysfunction

    PubMed Central

    Chapman, J.; Soloveichick, L.; Shavit, S.; Shoenfeld, Y.; Korczyn, A. D.

    2005-01-01

    Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (>50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of β2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of β2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through β2-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters. PMID:16295522

  15. Tyrphostin inhibition of ATP-stimulated DNA synthesis, cell proliferation and fos-protein expression in vascular smooth muscle cells.

    PubMed Central

    Erlinge, D.; Heilig, M.; Edvinsson, L.

    1996-01-01

    1. We and others have shown that extracellular ATP (adenosine triphosphate), released from sympathetic nerves and platelets, stimulates growth of vascular smooth muscle cells (SMC). To study the importance of tyrosine kinases for ATP-mediated proliferation in vascular smooth muscle cells we used tyrphostins, a recently developed group of highly specific inhibitors of tyrosine kinases. 2. ATP induced a powerful concentration-dependent increase in DNA synthesis measured by [3H]-thymidine incorporation in rat aorta SMC (RASMC) and an increase in total cell number after 72 h of incubation as measured by an enzymatic cell proliferation assay. Tyrphostin 25 (10(-5) M) had no effect per se on basal DNA synthesis but reduced ATP-stimulated DNA synthesis and increase in cell number in a dose-dependent manner. Higher concentrations of ATP could not reverse the inhibitory effect of tyrphostin 25. The potency of several (six) other tyrphostins was also examined and found to be slightly greater than tyrphostin 25 with equal efficacy. 3. When RASMC were incubated with 10(-5) M ATP for 2 h, nearly all of the cells (87 +/- 5%) were intensely stained with an antibody to the Fos protein while in the controls only 1 +/- 2% of the cells were weakly stained. Tyrphostin 25 greatly reduced the Fos-protein staining (14 +/- 2%). 4. ATP induced a concentration-dependent increase in 45Ca(2+)-influx and formation of inositol phosphates (IPtotal) in RASMC. These effects were not inhibited by tyrphostin 25. 5. Tyrphostin 25 did not alter ATP-induced contraction in ring segments of rat aorta. 6. In conclusion, tyrphostin 25 inhibited ATP-induced DNA synthesis, cell proliferation and Fos-protein expression, but not ATP-induced 45Ca(2+)-influx, inositolphosphate-production or vasoconstriction. This indicates that the mitogenic effect of ATP on vascular smooth muscle cells is dependent on tyrosine kinases in contrast to the contractile effect of ATP in blood vessels. Images Figure 2 PMID:8799578

  16. ATP synthase subunit-β down-regulation aggravates diabetic nephropathy

    PubMed Central

    Guan, Siao-Syun; Sheu, Meei-Ling; Wu, Cheng-Tien; Chiang, Chih-Kang; Liu, Shing-Hwa

    2015-01-01

    In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition. PMID:26449648

  17. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    PubMed

    Ippolito, Joseph E; Brandenburg, Matthew W; Ge, Xia; Crowley, Jan R; Kirmess, Kristopher M; Som, Avik; D'Avignon, D Andre; Arbeit, Jeffrey M; Achilefu, Samuel; Yarasheski, Kevin E; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer. PMID:27438712

  18. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide

    PubMed Central

    Ippolito, Joseph E.; Brandenburg, Matthew W.; Ge, Xia; Crowley, Jan R.; Kirmess, Kristopher M.; Som, Avik; D’Avignon, D. Andre; Arbeit, Jeffrey M.; Achilefu, Samuel; Yarasheski, Kevin E.; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer. PMID:27438712

  19. Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide.

    PubMed

    Ippolito, Joseph E; Brandenburg, Matthew W; Ge, Xia; Crowley, Jan R; Kirmess, Kristopher M; Som, Avik; D'Avignon, D Andre; Arbeit, Jeffrey M; Achilefu, Samuel; Yarasheski, Kevin E; Milbrandt, Jeffrey

    2016-01-01

    Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.

  20. 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.

  1. Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.

    PubMed

    Zouridakis, Marios; Zisimopoulou, Paraskevi; Eliopoulos, Elias; Poulas, Konstantinos; Tzartos, Socrates J

    2009-02-01

    In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved (125)I-alpha-bungarotoxin-binding affinity (K(d)=24 nM) compared to the wild-type-ECD (K(d)=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, d-tubocurarine or nicotine (K(i) of 21.5 nM, 127 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha7-ECD mutant for structural studies, useful for the rational drug design to

  2. Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in Ca

  3. Extracellular calcium sensing and extracellular calcium signaling

    NASA Technical Reports Server (NTRS)

    Brown, E. M.; MacLeod, R. J.; O'Malley, B. W. (Principal Investigator)

    2001-01-01

    The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others

  4. Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells

    PubMed Central

    Kim, Jeung-Il; Kim, Hye-Young; Kim, Sun-Mi; Lee, Sae-A; Son, Yong-Hae; Eo, Seong-Kug; Rhim, Byung-Yong

    2011-01-01

    To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+, an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD+- and NADP+-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process. PMID:21461238

  5. ATP release and contraction mediated by different P2-receptor subtypes in guinea-pig ileal smooth muscle

    PubMed Central

    Matsuo, Katsuichi; Katsuragi, Takeshi; Fujiki, Sono; Sato, Chiemi; Furukawa, Tatsuo

    1997-01-01

    The present study was addressed to clarify the subtypes of P2-purinoceptor involved in ATP release and contraction evoked by α,β-methylene ATP (α,β-mATP) and other P2-agonists in guinea-pig ileum.α,β-mATP 100 μM produced a transient and steep contraction followed by ATP release from tissue segments. These maximum responses appeared with different time-courses and their ED50 values were 5 and 25 μM, respectively. The maximum release of ATP by α,β-mATP was markedly reduced by 250 μM suramin, 30 μM pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (PPADS) and 30 μM reactive blue 2 (RB-2), P2-receptor antagonists. However, the contractile response was inhibited by suramin, tetrodotoxin and atropine, but not by PPADS and RB-2.Although the contraction caused by α,β-mATP was strongly diminished by Ca2+-removal and nifedipine, and also by tetrodotoxin and atropine at 0.3 μM, the release of ATP was virtually unaffected by these procedures.UTP, β,γ-methylene ATP (β,γ-mATP) and ADP at 100 μM elicited a moderate release of ATP. The release caused by UTP was virtually unaffected by RB-2. However, these P2-agonists failed to elicit a contraction of the segment.The potency order of all the agonists tested for the release of ATP was α,β-mATP>UTP>β,γ-mATP>ADP.In superfusion experiments with cultured smooth muscle cells from the ileum, α,β-mATP (100 μM) enhanced the release of ATP 5 fold above the basal value. This evoked release was inhibited by RB-2.These findings suggest that ATP release and contraction induced by P2-agonists such as α,β-mATP in the guinea-pig ileum result mainly from stimulation of different P2-purinoceptors, P2Y-like purinoceptors on the smooth muscles and, probably, P2X-purinoceptors on cholinergic nerve terminals, respectively. However, the ATP release may also be mediated, in part, by P2U-receptors, because UTP caused RB-2-insensitive ATP release. PMID:9283712

  6. Activation of trimeric P2X2 receptors by fewer than three ATP molecules.

    PubMed

    Stelmashenko, Olga; Lalo, Ulyana; Yang, Yue; Bragg, Laricia; North, R Alan; Compan, Vincent

    2012-10-01

    P2X receptors are trimeric membrane proteins. When they bind extracellular ATP, a conformational change occurs that opens a transmembrane ion channel. The ATP-binding pocket is formed in a cleft between two subunits, and a critical amino acid residue for ATP contact is Lys⁶⁹ (P2X2 numbering). In the present work, we sought to determine whether the binding of fewer than three ATP molecules could open the ion channel. We expressed eight concatenated cDNAs in human embryonic kidney cells, which encoded three serially joined, epitope-tagged, subunits with either Lys or Ala at position 69 (denoted as KKK, KKA, KAK, AKK, KAA, AKA, AAK, and AAA). Western blotting of surface-biotinylated proteins indicated that breakdown of concatemers to individual subunits was minimal. Recording of membrane currents in response to ATP (whole cell and excised outside-out patch) showed that all formed functional channels except AAK, AKA, and AAA. There was no difference in the kinetics of activation and deactivation among KKK, KKA, KAK, and AKK channels, and amplitude of the unitary conductances was in all cases not different from that found after expression of a single wild-type subunit. Currents through KKA and KAK receptors were larger than those observed for AKK receptors. The results indicate that trimeric P2X receptors containing only two intact binding sites can be readily activated by ATP.

  7. Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

    PubMed Central

    Genetos, Damian C.; Geist, Derik J.; Dawei, Liu; Donahue, Henry J.; Duncan, Randall L.

    2010-01-01

    ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell via L-type voltage-sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release. Introduction Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. Methods MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, then subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC) and vesicular formation were added prior to shear and maintained in the flow medium for the duration of the experiment. Results and Conclusions Fluid shear produced a transient increase in ATP release compared to static MC3T3-E1 cells (59.8±15.7nM vs. 6.2±1.8nM, respectively), peaking within 1 min of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca2+ -dependent ATP release in response to shear appeared to result from vesicular release, and not through gap hemichannels, since vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP

  8. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

  9. Energy-dependent transformation of the catalytic activities of the mitochondrial F0 x F1-ATP synthase.

    PubMed

    Galkin, M A; Vinogradov, A D

    1999-04-01

    The ADP(Mg2+)-deactivated, azide-trapped F0 x F1-ATPase of coupled submitochondrial particles is capable of ATP synthesis being incapable of ATP hydrolysis and ATP-dependent delta muH+ generation [FEBS Lett. (1995) 366, 29-32]. This puzzling phenomenon was studied further. No ATPase activity of the submitochondrial particles catalyzing succinate-supported oxidative phosphorylation in the presence of azide was observed when ATP was added to the assay mixture after an uncoupler. Rapid ATP hydrolysis was detected in the same system when ATP followed by an uncoupler was added. Less than 5% of the original ATPase activity was seen when the reaction (assayed with ATP-regenerating system) was initiated by the addition of ATP to the azide-trapped coupled particles oxidizing succinate either in the presence or in the absence of the uncoupler. High ATP hydrolytic activity was revealed when the reaction was started by the simultaneous addition of the ATP plus uncoupler to the particles generating delta muH+. The energy-dependent conversion of the enzyme into latent uncoupler-activated ATPase was prevented by free ADP (Ki approximately 20 microM) and was greatly enhanced after multiple turnovers in oxidative phosphorylation. The results suggest that the catalytic properties of F0 x F1 are delta muH+-dependent which is in accord with our hypothesis on different conformational states of the enzyme participating in ATP synthesis or hydrolysis.

  10. A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

    PubMed

    Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

    2015-12-15

    The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment.

  11. Tunable Fluorescent Silica-Coated Carbon Dots: A Synergistic Effect for Enhancing the Fluorescence Sensing of Extracellular Cu²⁺ in Rat Brain.

    PubMed

    Lin, Yuqing; Wang, Chao; Li, Linbo; Wang, Hao; Liu, Kangyu; Wang, Keqing; Li, Bo

    2015-12-16

    Carbon quantum dots (CDs) combined with self-assembly strategy have created an innovative way to fabricate novel hybrids for biological analysis. This study demonstrates a new fluorescence platform with enhanced selectivity for copper ion sensing in the striatum of the rat brain following the cerebral calm/sepsis process. Here, the fabrication of silica-coated CDs probes is based on the efficient hybridization of APTES which act as a precursor of organosilane self-assembly, with CDs to form silica-coated CDs probes. The fluorescent properties including intensity, fluorescence quantum yield, excitation-independent region, and red/blue shift of the emission wavelength of the probe are tunable through reliable regulation of the ratio of CDs and APTES, realizing selectivity and sensitivity-oriented Cu(2+) sensing. The as-prepared probes (i.e., 3.33% APTES-0.9 mg mL(-1) CDs probe) show a synergistic amplification effect of CDs and APTES on enhancing the fluorescence signal of Cu(2+) detection through fluorescent self-quenching. The underlying mechanism can be ascribed to the stronger interaction including chelation and electrostatic attraction between Cu(2+) and N and O atoms-containing as well as negatively charged silica-coated CDs than other interference. Interestingly, colorimetric assay and Tyndall effect can be observed and applied to directly distinguish the concentration of Cu(2+) by the naked eye. The proposed fluorescent platform here has been successfully applied to monitor the alteration of striatum Cu(2+) in rat brain during the cerebral calm/sepsis process. The versatile properties of the probe provide a new and effective fluorescent platform for the sensing method in vivo sampled from the rat brain. PMID:26592139

  12. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    PubMed

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-01

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

  13. Permeation of both cations and anions through a single class of ATP- activated ion channels in developing chick skeletal muscle

    PubMed Central

    1990-01-01

    Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS. PMID:1692581

  14. Mechanical modulation of ATP-binding affinity of V1-ATPase.

    PubMed

    Tirtom, Naciye Esma; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

    2013-01-01

    V(1)-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F(1)-ATPase (the closest relative of V(1)-ATPase evolutionarily), the role of ATP binding for V(1)-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V(1)-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V(1)-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V(1)-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k(on)) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k(off)) was exponentially reduced. The angle dependence of the k(off) of V(1)-ATPase was significantly smaller than that of F(1)-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V(1)-ATPase. When V(1)-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k(on) was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V(1)-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.

  15. α-Synuclein forms non-selective cation channels and stimulates ATP-sensitive potassium channels in hippocampal neurons

    PubMed Central

    Mironov, Sergej L

    2015-01-01

    In Parkinson's disease and several other neurodegenerative diseases, the protein α-synuclein (αS) is produced within neurons and accumulates in the extracellular fluid. Several mechanisms of αS action are proposed, one of which is the formation of cation-permeable pores that may mediate toxicity. αS induces non-selective cation channels in lipid bilayers, but whether this occurs in living neurons and which properties the channels possess have not yet been examined. In this study the properties of αS channels in dissociated hippocampal neurons are documented. In cell-attached recordings the incorporation of αS into membranes was driven by applied negative potentials. These channels exhibited multiple levels of conductance (30, 70 and 120 pS at −100 mV) and inward rectification. The persistent activity of αS channels induced local changes in intracellular Na+ and Ca2+, depolarized neurons and augmented bursting activity. αS channels formed by adding αS to the intracellular membrane in inside-out patches exhibited outward rectification. αS channels were equally permeable to Na+, K+ and Ca2+. These channels were also observed in neurons transfected with wild-type or mutant A53T αS, and after extracellular application of wild-type or mutant A53T αS proteins. Opening of αS channels stimulated opening of ATP-sensitive K+ (KATP ) channels and did not interfere with the activity of delayed rectifier K+ channels. The properties of αS channels in neuronal membranes suggest stronger toxicity of extracellularly applied αS than intracellular αS. Enhancement of neuronal excitability and distortions in ion homeostasis may underlie the toxic effects of αS that can be dampened by KATP channels. PMID:25556793

  16. The Signaling Mechanism of Contraction Induced by ATP and UTP in Feline Esophageal Smooth Muscle Cells

    PubMed Central

    Kwon, Tae Hoon; Jung, Hyunwoo; Cho, Eun Jeong; Jeong, Ji Hoon; Sohn, Uy Dong

    2015-01-01

    P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or PLCβ3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi3 and G q proteins, which activate PLCβ1 and PLCβ3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction. PMID:26013385

  17. Variation potential-induced photosynthetic and respiratory changes increase ATP content in pea leaves.

    PubMed

    Surova, Lyubov; Sherstneva, Oksana; Vodeneev, Vladimir; Katicheva, Lyubov; Semina, Maria; Sukhov, Vladimir

    2016-09-01

    Local damage induces a physiological response in higher plants by means of generation and propagation of variation potential (VP). The response includes changes in photosynthesis and respiration. The aim of the present study was to investigate the effect of these changes on adenosine triphosphate (ATP) content in pea leaves. VP was induced by local heating of the first mature leaf and registered using extracellular and intracellular electrodes. Photosynthesis and respiration were measured using Dual-PAM-100 and GFS-3000. ATP content was determined using a bioluminescence-based ATP determination kit. Two non-stimulated leaves (second and fourth) were investigated. We showed that heating induced VP that propagated into the second mature leaf, but only a slight electrical reaction was registered in the fourth mature leaf. VP-induced inactivation of photosynthesis developed in the second leaf and included two stages: short- and long-term inactivation. Local heating also caused a two-stage increase in ATP content in the second leaf, which was connected with the photosynthetic responses. Changes in photosynthesis and ATP content were not observed in the fourth leaf. The effect of VP on respiration was investigated under dark conditions. We found that variation potential induced short-term activation of respiration in the second leaf. Local heating induced ATP content increase which included only one stage under dark conditions. Changes in ATP and respiration were absent in the fourth leaf under dark conditions. Thus, VP-induced photosynthetic and respiratory changes are likely to increase ATP content in pea leaves. PMID:27450494

  18. Variation potential-induced photosynthetic and respiratory changes increase ATP content in pea leaves.

    PubMed

    Surova, Lyubov; Sherstneva, Oksana; Vodeneev, Vladimir; Katicheva, Lyubov; Semina, Maria; Sukhov, Vladimir

    2016-09-01

    Local damage induces a physiological response in higher plants by means of generation and propagation of variation potential (VP). The response includes changes in photosynthesis and respiration. The aim of the present study was to investigate the effect of these changes on adenosine triphosphate (ATP) content in pea leaves. VP was induced by local heating of the first mature leaf and registered using extracellular and intracellular electrodes. Photosynthesis and respiration were measured using Dual-PAM-100 and GFS-3000. ATP content was determined using a bioluminescence-based ATP determination kit. Two non-stimulated leaves (second and fourth) were investigated. We showed that heating induced VP that propagated into the second mature leaf, but only a slight electrical reaction was registered in the fourth mature leaf. VP-induced inactivation of photosynthesis developed in the second leaf and included two stages: short- and long-term inactivation. Local heating also caused a two-stage increase in ATP content in the second leaf, which was connected with the photosynthetic responses. Changes in photosynthesis and ATP content were not observed in the fourth leaf. The effect of VP on respiration was investigated under dark conditions. We found that variation potential induced short-term activation of respiration in the second leaf. Local heating induced ATP content increase which included only one stage under dark conditions. Changes in ATP and respiration were absent in the fourth leaf under dark conditions. Thus, VP-induced photosynthetic and respiratory changes are likely to increase ATP content in pea leaves.

  19. A Tetrahymena Hsp90 co-chaperone promotes siRNA loading by ATP-dependent and ATP-independent mechanisms.

    PubMed

    Woehrer, Sophie L; Aronica, Lucia; Suhren, Jan H; Busch, Clara Jana-Lui; Noto, Tomoko; Mochizuki, Kazufumi

    2015-02-12

    The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the ATP-independent activity. Both activities facilitate siRNA loading by counteracting the Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a physiological role in Tetrahymena.

  20. On the ATP binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2016-04-01

    F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells.

  1. Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

    SciTech Connect

    Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A.; Xing, Yongna

    2013-10-08

    Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.

  2. ATP-induced noncooperative thermal unfolding of hen lysozyme

    SciTech Connect

    Liu, Honglin; Yin, Peidong; He, Shengnan; Sun, Zhihu; Tao, Ye; Huang, Yan; Zhuang, Hao; Zhang, Guobin; Wei, Shiqiang

    2010-07-02

    To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg{sup 2+}-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the {beta}-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich {alpha}-helix and less {beta}-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric {beta}-sheet enriched intermediate.

  3. Treatment of heterotopic ossification through remote ATP hydrolysis.

    PubMed

    Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

    2014-09-24

    Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. PMID:25253675

  4. 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. PMID:25191981

  5. IV ATP Potentiates Midazolam Sedation as Assessed by Bispectral Index

    PubMed Central

    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. PMID:25191981

  6. Interaction between ATP, metal ions, glycine, and several minerals

    NASA Technical Reports Server (NTRS)

    Rishpon, J.; Ohara, P. J.; Lawless, J. G.; Lahav, N.

    1982-01-01

    Interactions between ATP, glycine and montmorillonite and kaolinite clay minerals in the presence of various metal cations are investigated. The adsorption of adenine nucleotides on clays and Al(OH)3 was measured as a function of pH, and glycine condensation was followed in the presence of ATP, ZnCl2, MgCl2 and either kaolinite or montmorillonite. The amounts of ATP and ADP adsorbed are found to decrease with increasing Ph, and to be considerably enhanced in experiments with Mg(2+)- and Zn(2+)-montmorillonite with respect to Na(+)-montmorillonite. The effects of divalent cations are less marked in kaolinite. Results for Al(OH)3 show the importance of adsorption at clay platelet edges at high pH. The decomposition of ATP during drying at high temperature is observed to be inhibited by small amounts of clay, vacuum, or Mg(2+) or Zn(2+) ions, and to be accompanied by peptide formation in the presence of glycine. Results suggest the importance of Zn(2+) and Mg(2+) in chemical evolution.

  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. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  9. ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

    PubMed Central

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-01-01

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

  10. Sodium pump-mediated ATP:ADP exchange. The sided effects of sodium and potassium ions

    PubMed Central

    1982-01-01

    Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump. PMID:6294224

  11. ATP released by electrical stimuli elicits calcium transients and gene expression in skeletal muscle.

    PubMed

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-12-11

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca(2+) homeostasis and muscle physiology. PMID:19822518

  12. Nuclear genetic defects of mitochondrial ATP synthase.

    PubMed

    Hejzlarová, K; Mráček, T; Vrbacký, M; Kaplanová, V; Karbanová, V; Nůsková, H; Pecina, P; Houštěk, J

    2014-01-01

    Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting as early-onset mitochondrial encephalo-cardiomyopathies. Up to now, mutations in four nuclear genes were associated with isolated deficiency of ATP synthase. Two of them, ATP5A1 and ATP5E encode enzyme's structural subunits alpha and epsilon, respectively, while the other two ATPAF2 and TMEM70 encode specific ancillary factors that facilitate the biogenesis of ATP synthase. All these defects share a similar biochemical phenotype with pronounced decrease in the content of fully assembled and functional ATP synthase complex. However, substantial differences can be found in their frequency, molecular mechanism of pathogenesis, clinical manifestation as well as the course of the disease progression. While for TMEM70 the number of reported patients as well as spectrum of the mutations is steadily increasing, mutations in ATP5A1, ATP5E and ATPAF2 genes are very rare. Apparently, TMEM70 gene is highly prone to mutagenesis and this type of a rare mitochondrial disease has a rather frequent incidence. Here we present overview of individual reported cases of nuclear mutations in ATP synthase and discuss, how their analysis can improve our understanding of the enzyme biogenesis.

  13. Action of ATP on ventricular automaticity.

    PubMed

    Stark, G; Domanowits, H; Sterz, F; Stark, U; Bachernegg, M; Kickenweiz, E; Decrinis, M; Laggner, A N; Tritthart, H A

    1994-11-01

    ATP is an effective treatment of supraventricular tachycardia when the atrioventricular (AV) node is part of the reentrant circuit. However, the lower a pace-maker in the pacemaker hierarchy, the more sensitive it is to adenosine. Therefore, we investigated the effects of ATP on ventricular automaticity in in vivo and in vitro conditions. Wide and narrow QRS complex tachycardia in 46 patients was treated with 6, 12, and 18 mg ATP as sequential intravenous (i.v.) bolus. ATP terminated tachycardias in 67%. Bolus infusion ATP caused < or = 6.4-s asystole that was self-limited. Perfusion of isolated spontaneously beating guinea pig heart with 100 microM ATP completely suppressed ventricular automaticity. After ATP-infusion was discontinued, the first ventricular beat was evident after 3.1 +/- 0.9 s and sinus node activity recovered with a time constant of 3.0 +/- 1.1 s. Because sinus node and ventricular automaticity recovered within seconds after ATP infusion was discontinued in vitro, recovery in vivo is also likely to be determined by the short half-life (+1/2) of ATP. PMID:7532751

  14. Neuropeptide Y and extracellular signal-regulated kinase mediate injury-induced neuroregeneration in mouse olfactory epithelium

    PubMed Central

    Jia, Cuihong; Hegg, Colleen Cosgrove

    2011-01-01

    In the olfactory epithelium (OE), injury induces ATP release, and subsequent activation of P2 purinergic receptors by ATP promotes neuroregeneration by increasing basal progenitor cell proliferation. The molecular mechanisms underlying ATP-induced increases in OE neuroregeneration have not been established. In the present study, the roles of neuroproliferative factors neuropeptide Y (NPY) and fibroblast growth factor2 (FGF2), and p44/42 extracellular signal-regulated kinase (ERK) on ATP-mediated increases of neuroregeneration in the OE were investigated. ATP increased basal progenitor cell proliferation in the OE via activation of P2 purinergic receptors in vitro and in vivo as monitored by incorporation of 5′-ethynyl-2′-deoxyuridine, a thymidine analog, into DNA, and proliferating cell nuclear antigen (PCNA) protein levels. ATP induced p44/42 ERK activation in globose basal cells (GBC) but not horizontal basal cells (HBC). ATP differentially regulated p44/42 ERK over time in the OE both in vitro and in vivo with transient inhibition (5–15 min) followed by activation (30 min – 1 hr) of p44/42 ERK. In addition, ATP indirectly activated p44/42 ERK in the OE via ATP-induced NPY release and subsequent activation of NPY Y1 receptors in the basal cells. There were no synergistic effects of ATP and NPY or FGF2 on OE neuroregeneration. These data clearly have implications for the pharmacological modulation of neuroregeneration in the olfactory epithelium. PMID:22154958

  15. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

    PubMed Central

    Xing, Shu; Grol, Matthew W.; Grutter, Peter H.; Dixon, S. Jeffrey; Komarova, Svetlana V.

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP. PMID:27468270

  16. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    PubMed

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP. PMID:27468270

  17. Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

    PubMed

    Xing, Shu; Grol, Matthew W; Grutter, Peter H; Dixon, S Jeffrey; Komarova, Svetlana V

    2016-01-01

    Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

  18. (31)P-MRS of healthy human brain: ATP synthesis, metabolite concentrations, pH, and T1 relaxation times.

    PubMed

    Ren, Jimin; Sherry, A Dean; Malloy, Craig R

    2015-11-01

    The conventional method for measuring brain ATP synthesis is (31)P saturation transfer (ST), a technique typically dependent on prolonged pre-saturation with γ-ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ-ATP magnetization in the absence of B1 field following co-inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the Pi → γ-ATP reaction is 0.21 ± 0.04 s(-1) and the ATP synthesis rate is 9.9 ± 2.1 mmol min(-1)  kg(-1) in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable (31)P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T1 shorter than that of monophosphates (Pi, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the (31)P-(31)P dipolar mechanism in T1 relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T1 for intracellular Pi relative to extracellular Pi, attributed to the modulation by the intracellular phosphoryl exchange reaction Pi ↔ γ-ATP. The sufficiently separated intra- and extracellular Pi signals also permit the distinction of pH between intra- and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative (31)P MRS in combination with ATP synthesis, pH, and T1 relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases

  19. (31)P-MRS of healthy human brain: ATP synthesis, metabolite concentrations, pH, and T1 relaxation times.

    PubMed

    Ren, Jimin; Sherry, A Dean; Malloy, Craig R

    2015-11-01

    The conventional method for measuring brain ATP synthesis is (31)P saturation transfer (ST), a technique typically dependent on prolonged pre-saturation with γ-ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ-ATP magnetization in the absence of B1 field following co-inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the Pi → γ-ATP reaction is 0.21 ± 0.04 s(-1) and the ATP synthesis rate is 9.9 ± 2.1 mmol min(-1)  kg(-1) in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable (31)P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T1 shorter than that of monophosphates (Pi, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the (31)P-(31)P dipolar mechanism in T1 relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T1 for intracellular Pi relative to extracellular Pi, attributed to the modulation by the intracellular phosphoryl exchange reaction Pi ↔ γ-ATP. The sufficiently separated intra- and extracellular Pi signals also permit the distinction of pH between intra- and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative (31)P MRS in combination with ATP synthesis, pH, and T1 relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases.

  20. ATP/ADP Ratio, the Missed Connection between Mitochondria and the Warburg Effect

    PubMed Central

    Maldonado, Eduardo N.; Lemasters, John J.

    2014-01-01

    Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives release of ATP−4 from the matrix in exchange for ADP−3 in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis. PMID:25229666

  1. Extracellular nucleotide and nucleoside signaling in vascular and blood disease

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Riegel, Ann-Kathrin

    2014-01-01

    Nucleotides and nucleosides—such as adenosine triphosphate (ATP) and adenosine—are famous for their intracellular roles as building blocks for the genetic code or cellular energy currencies. In contrast, their function in the extracellular space is different. Here, they are primarily known as signaling molecules via activation of purinergic receptors, classified as P1 receptors for adenosine or P2 receptors for ATP. Because extracellular ATP is rapidly converted to adenosine by ectonucleotidase, nucleotide-phosphohydrolysis is important for controlling the balance between P2 and P1 signaling. Gene-targeted mice for P1, P2 receptors, or ectonucleotidase exhibit only very mild phenotypic manifestations at baseline. However, they demonstrate alterations in disease susceptibilities when exposed to a variety of vascular or blood diseases. Examples of phenotypic manifestations include vascular barrier dysfunction, graft-vs-host disease, platelet activation, ischemia, and reperfusion injury or sickle cell disease. Many of these studies highlight that purinergic signaling events can be targeted therapeutically. PMID:25001468

  2. TRPC channel activation by extracellular thioredoxin

    PubMed Central

    Xu, Shang-Zhong; Sukumar, Piruthivi; Zeng, Fanning; Li, Jing; Jairaman, Amit; English, Anne; Naylor, Jacqueline; Ciurtin, Coziana; Majeed, Yasser; Milligan, Carol J; Bahnasi, Yahya M; AL-Shawaf, Eman; Porter, Karen E; Jiang, Lin-Hua; Emery, Paul; Sivaprasadarao, Asipu; Beech, David J

    2009-01-01

    Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor1,2. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these novel channels. Here we show activation of TRPC5 homomultimeric and TRPC5-TRPC1 heteromultimeric channels3-5 by extracellular reduced thioredoxin acting by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown6-9. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis8,10-12, an inflammatory joint disease disabling millions of people world-wide13. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and blockade of the channels enhances secretory activity and prevents suppression of secretion by thioredoxin. The data suggest a novel ion channel activation mechanism that couples extracellular thioredoxin to cell function. PMID:18172497

  3. Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice.

    PubMed

    Seino, S; Iwanaga, T; Nagashima, K; Miki, T

    2000-03-01

    The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K(ATP

  4. 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.

  5. Role of Ca2+ in responses of airway epithelia to Pseudomonas aeruginosa, flagellin, ATP, and thapsigargin.

    PubMed

    Fu, Zhu; Bettega, Kelly; Carroll, Susheela; Buchholz, Kerry R; Machen, Terry E

    2007-01-01

    Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased [Ca](i) but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca](i)) or Ca(2+)-free solution reduced increases in [Ca](i) due to ATP or thapsigargin and also reduced NF-kappaB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-kappaB-binding site was critical for activation by all agonists and for activation by [Ca](i). Thus increased [Ca](i) was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased [Ca](i) was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca](i) synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca](i) reduced these responses. Thus [Ca](i) plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in [Ca](i). PMID:16963531

  6. Insulin-stimulated phosphorylation of ATP-citrate lyase in isolated hepatocytes. Stoichiometry and relation to the phosphoenzyme intermediate.

    PubMed

    Alexander, M C; Palmer, J L; Pointer, R H; Kowaloff, E M; Koumjian, L L; Avruch, J

    1982-02-25

    We have estimated the insulin-stimulated phosphorylation of ATP-citrate lyase by two methods. Isolated hepatocytes incorporate extracellular 32P into [gamma-35P] ATP and immunoprecipitated ATP-citrate lyase to steady state levels by 1 h. The content of acid-stable 32P in hepatocyte ATP-citrate lyase at steady state is 0.33 +/- 0.038 mol of P/mol (tetrameric) holoenzyme. Insulin (1 milliunit/ml) increases the 32P content of immunoprecipitated lyase 2- to 3-fold in 10 min. Over 90% of acid-stable 32P on lyase is 32P-serine in enzyme isolated from both control and insulin-treated cells. ATP-citrate lyase isolated from hepatocytes contains 0.95 +/- 0.1 mol of alkali-labile phosphate/mol of holoenzyme. Insulin treatment of hepatocytes (1 milliunit/ml for 10 min) increases the alkali-labile P content by 45%. Evidence is presented which indicates that the insulin-stimulated phosphorylation does not arise by intramolecular migration from the catalytic phosphoenzyme intermediate. These observations support the conclusion that insulin-stimulated phosphorylation of ATP-citrate lyase is mediated either by an insulin-induced increase in the activity of lyase kinase and/or decrease in a lyase phosphatase. The functional role of the substoichiometric phosphorylation of ATP-citrate lyase remains unknown.

  7. Thermodynamics of proton transport coupled ATP synthesis.

    PubMed

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase.

  8. Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

    PubMed

    Plattner, Helmut; Verkhratsky, Alexei

    2016-08-01

    From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally

  9. Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

    PubMed

    Plattner, Helmut; Verkhratsky, Alexei

    2016-08-01

    From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally

  10. Inhibition of Connexin 43 Hemichannel-Mediated ATP Release Attenuates Early Inflammation During the Foreign Body Response

    PubMed Central

    Calder, Bennett W.; Rhett, Joshua Matthew; Bainbridge, Heather; Fann, Stephen A.; Gourdie, Robert G.

    2015-01-01

    Background: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction. Methods: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis. Results: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle. Conclusions: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory

  11. Effect of Intramuscular Protons, Lactate, and ATP on Muscle Hyperalgesia in Rats.

    PubMed

    Gregory, Nicholas S; Whitley, Phillip E; Sluka, Kathleen A

    2015-01-01

    Chronic muscle pain is a significant health problem leading to disability[1]. Muscle fatigue can exacerbate muscle pain. Metabolites, including ATP, lactate, and protons, are released during fatiguing exercise and produce pain in humans. These substances directly activate purinergic (P2X) and acid sensing ion channels (ASICs) on muscle nociceptors, and when combined, produce a greater increase in neuron firing than when given alone. Whether the enhanced effect of combining protons, lactate, and ATP is the sum of individual effects (additive) or more than the sum of individual effects (synergistic) is unknown. Using a rat model of muscle nociceptive behavior, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle nociceptive behavior. We also tested a non-hydrolyzable form of ATP (α,β-meATP) alone and in combination with lactate and acidic pH. Surprisingly, we found no dose-dependent effect on muscle nociceptive behavior for protons, lactate, or ATP when given alone. We similarly found no effect after application of each two-metabolite combination. Only pH 4 saline and α,β-meATP produced hyperalgesia when given alone. When all 3 substances were combined, however, ATP (2.4μm), lactate (10mM), and acidic pH (pH 6.0) produced an enhanced effect greater than the sum of the effects of the individual components, i.e. synergism. α,β me ATP (3nmol), on the other hand, showed no enhanced effects when combined with lactate (10mM) or acidic pH (pH 6.0), i.e. additive. These data suggest that combining fatigue metabolites in muscle produces a synergistic effect on muscle nociception.

  12. TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

    PubMed

    Kwon, Hyuck Joon

    2012-08-10

    Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels. This study suggests that Ca(2+)-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis.

  13. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity

    PubMed Central

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-01-01

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut−/−) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut−/− mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut−/− mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut−/− mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

  14. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

    PubMed

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-10-21

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

  15. 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.

  16. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

    PubMed Central

    Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

    2015-01-01

    Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

  17. 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. PMID:26939579

  18. Role of glycogenolysis in stimulation of ATP release from cultured mouse astrocytes by transmitters and high K+ concentrations

    PubMed Central

    Xu, Junnan; Song, Dan; Bai, Qiufang; Zhou, Lijun; Cai, Liping; Hertz, Leif; Peng, Liang

    2013-01-01

    This study investigates the role of glycogenolysis in stimulated release of ATP as a transmitter from astrocytes. Within the last 20 years our understanding of brain glycogenolysis has changed from it being a relatively uninteresting process to being a driving force for essential brain functions like production of transmitter glutamate and homoeostasis of potassium ions (K+) after their release from excited neurons. Simultaneously, the importance of astrocytic handling of adenosine, its phosphorylation to ATP and release of some astrocytic ATP, located in vesicles, as an important transmitter has also become to be realized. Among the procedures stimulating Ca2+-dependent release of vesicular ATP are exposure to such transmitters as glutamate and adenosine, which raise intra-astrocytic Ca2+ concentration, or increase of extracellular K+ to a depolarizing level that opens astrocytic L-channels for Ca2+ and thereby also increase intra-astrocytic Ca2+ concentration, a prerequisite for glycogenolysis. The present study has confirmed and quantitated stimulated ATP release from well differentiated astrocyte cultures by glutamate, adenosine or elevated extracellular K+ concentrations, measured by a luciferin/luciferase reaction. It has also shown that this release is virtually abolished by an inhibitor of glycogenolysis as well as by inhibitors of transmitter-mediated signaling or of L-channel opening by elevated K+ concentrations. PMID:24328680

  19. Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals.

    PubMed

    Hovater, Michael B; Olteanu, Dragos; Hanson, Elizabeth L; Cheng, Nai-Lin; Siroky, Brian; Fintha, Attila; Komlosi, Peter; Liu, Wen; Satlin, Lisa M; Bell, P Darwin; Yoder, Bradley K; Schwiebert, Erik M

    2008-06-01

    mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases. PMID:18368523

  20. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    PubMed Central

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G.; Møller, Lisbeth B.

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6–8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  1. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures.

    PubMed

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz; Jensen, Thomas G; Møller, Lisbeth B

    2016-01-01

    Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD) or the rare autosomal disorder Wilson disease (WD), respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation. PMID:27587995

  2. Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

    PubMed

    Kim, Ha Won; Chan, Qilin; Afton, Scott E; Caruso, Joseph A; Lai, Barry; Weintraub, Neal L; Qin, Zhenyu

    2012-02-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

  3. Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds

    PubMed Central

    Kim, Ha Won; Chan, Qilin; Afton, Scott E.; Caruso, Joseph A.; Lai, Barry; Weintraub, Neal L.; Qin, Zhenyu

    2013-01-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound

  4. Farnesoid X receptor activation by chenodeoxycholic acid induces detoxifying enzymes through AMP-activated protein kinase and extracellular signal-regulated kinase 1/2-mediated phosphorylation of CCAAT/enhancer binding protein β.

    PubMed

    Noh, Kyoung; Kim, Young Mi; Kim, Young Woo; Kim, Sang Geon

    2011-08-01

    Farnesoid X receptor (FXR) regulates redox homeostasis and elicits a cytoprotective effect. CCAAT/enhancer binding protein-β (C/EBPβ) plays a role in regulating the expression of hepatocyte-specific genes and contributes to hepatocyte protection and liver regeneration. In view of the role of FXR in xenobiotic metabolism and hepatocyte survival, this study investigated the potential of FXR to activate C/EBPβ for the induction of detoxifying enzymes and the responsible regulatory pathway. Chenodeoxycholic acid (CDCA), a major component in bile acids, activates FXR. In HepG2 cells, CDCA treatment activated C/EBPβ, as shown by increases in its phosphorylation, nuclear accumulation, and expression. 3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl-)oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR ligand, had similar effects. In addition, CDCA enhanced luciferase gene transcription from the construct containing -1.65-kb GSTA2 promoter, which contained C/EBP response element (pGL-1651). Moreover, CDCA treatment activated AMP-activated protein kinase (AMPK), which led to extracellular signal-regulated kinase 1/2 (ERK1/2) activation, as evidenced by the results of experiments using a dominant-negative mutant of AMPKα and chemical inhibitor. The activation of ERK1/2 was responsible for the activating phosphorylation of C/EBPβ. FXR knockdown attenuated the ability of CDCA to activate AMPK and ERK1/2 and phosphorylate C/EBPβ. Consistently, enforced expression of FXR promoted the phosphorylation of AMPKα, ERK1/2, and C/EBPβ, verifying that C/EBPβ phosphorylation elicited by CDCA results from the activation of AMPK and ERK1/2 by FXR. In mice, CDCA treatment activated C/EBPβ with the induction of detoxifying enzymes in the liver. Our results demonstrate that CDCA induces antioxidant and xenobiotic-metabolizing enzymes by activating C/EBPβ through AMPK-dependent ERK1/2 pathway downstream of FXR.

  5. Intradermal administration of ATP augments methacholine-induced cutaneous vasodilation but not sweating in young males and females.

    PubMed

    Fujii, Naoto; Halili, Lyra; Singh, Maya Sarah; Meade, Robert D; Kenny, Glen P

    2015-10-15

    Acetylcholine released from cholinergic nerves is a key neurotransmitter contributing to heat stress-induced cutaneous vasodilation and sweating. Given that sympathetic cholinergic nerves also release ATP, ATP may play an important role in modulating cholinergic cutaneous vasodilation and sweating. However, the pattern of response may differ between males and females given reports of sex-related differences in the peripheral mechanisms governing these heat loss responses. Cutaneous vascular conductance (CVC, laser-Doppler perfusion units/mean arterial pressure) and sweat rate (ventilated capsule) were evaluated in 17 young adults (8 males, 9 females) at four intradermal microdialysis skin sites continuously perfused with: 1) lactated Ringer (Control), 2) 0.3 mM ATP, 3) 3 mM ATP, or 4) 30 mM ATP. At all skin sites, methacholine was coadministered in a concentration-dependent manner (0.0125, 0.25, 5, 100, 2,000 mM, each for 25 min). In both males and females, CVC was elevated with the lone infusion of 30 mM ATP (both P < 0.05), but not with 0.3 and 3 mM ATP compared with control (all P >0.27). However, 0.3 mM ATP induced a greater increase in CVC compared with control in response to 100 mM methacholine infusion in males (P < 0.05). In females, 0.3 mM ATP infusion resulted in a lower concentration of methacholine required to elicit a half-maximal response (EC50) (P < 0.05). In both males and females, methacholine-induced sweating was unaffected by any concentration of ATP (all P > 0.44). We demonstrate that ATP enhances cholinergic cutaneous vasodilation albeit the pattern of response differs between males and females. Furthermore, we show that ATP does not modulate cholinergic sweating. PMID:26290105

  6. The Rotary Mechanism of the ATP Synthase

    PubMed Central

    Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.

    2008-01-01

    The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. PMID:18515057

  7. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  8. Binding of ATP to the progesterone receptor.

    PubMed Central

    Moudgil, V K; Toft, D O

    1975-01-01

    The possible interaction of progesterone--receptor complexes with nucleotides was tested by affinity chromatography. The cytosol progesterone receptor from hen oviduct was partially purified by ammonium sulfate precipitation before use. When progesterone was bound