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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yun

quantification mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca 2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca 2+ signaling through gap junctions seems more effective.« less

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

Serotonergic regulation of distention-induced ATP release from the urothelium.

PubMed

Matsumoto-Miyai, Kazumasa; Yamada, Erika; Shinzawa, Eriko; Koyama, Yoshihisa; Shimada, Shoichi; Yoshizumi, Masaru; Kawatani, Masahito

2016-04-01

Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT 1D and 5-HT 4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH 2 O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT 1B/1D antagonist GR-127935 but not by the 5-HT 4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT 4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT 1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT 1D . Copyright © 2016 the American Physiological Society.

The potent inhibition of vapiprost, a novel thromboxane A2 receptor antagonist, on the secondary aggregation and ATP release of human platelets.

PubMed

Horie, S; Yamada, M; Satoh, M; Noritake, S; Hiraishi, S; Kizaki, K; Kurusu, O; Nakahara, T; Ishii, H; Kazama, M

1997-06-01

The inhibitory effects of vapiprost hydrochloride (vapiprost), a novel thromboxane A2 receptor antagonist, on platelet aggregation and ATP release were studied using platelet rich plasma (PRP) of humans, guinea pigs, rabbits and rats. In in vitro experiments with human platelet, vapiprost inhibited the aggregation and ATP release stimulated with U-46619, collagen or arachidonic acid (AA) at an IC50 of less than 2.1 x 10(-8) M. Vapiprost did not inhibit the primary aggregation or ATP release of human platelets stimulated with adenosine 5'-diphosphate (ADP), epinephrine (Epi) or platelet activating factor (PAF), but inhibited the secondary aggregation stimulated with those agonists at an IC50 of less than 1.3 x 10(-7) M. The sensitivity of platelets in various species of animals to vapiprost was in the following order: human > or = guinea pigs > rats > rabbits. In ex vivo experiments with guinea pigs which received a single oral dose of vapiprost, the agent demonstrated strong inhibition of ATP release from platelets stimulated with U-46619, collagen or AA at an ID50 of less than 25.8 micrograms/kg. These inhibitory effects were observed within 30 min and sustained for 24 h at a single dosage of 5 mg/kg of vapiprost. In AA-induced pulmonary infarction models of mice, the sudden death rates decreased significantly with the oral administration of 10 mg/kg or more of vapiprost. These results indicate that vapiprost effectively inhibits the secondary aggregation and ATP release of human platelets stimulated with various agonists, and that guinea pig and human platelets are similar in response to vapiprost. Furthermore, it was demonstrated in ex vivo experiments with guinea pigs that the inhibitory action of vapiprost appears rapidly and lasts for long periods.

Simultaneous analysis of vascular norepinephrine and ATP release using an integrated microfluidic system.

PubMed

Townsend, Alexandra D; Wilken, Gerald H; Mitchell, Kyle K; Martin, R Scott; Macarthur, Heather

2016-06-15

Sympathetic nerves are known to release three neurotransmitters: norepinephrine, ATP, and neuropeptide Y that play a role in controlling vascular tone. This paper focuses on the co-release of norepinephrine and ATP from the mesenteric arterial sympathetic nerves of the rat. In this paper, a quantification technique is described that allows simultaneous detection of norepinephrine and ATP in a near-real-time fashion from the isolated perfused mesenteric arterial bed of the rat. Simultaneous detection is enabled with 3-D printing technology, which is shown to help integrate the perfusate with different detection methods (norepinephrine by microchip-based amperometery and ATP by on-line chemiluminescence). Stimulated levels relative to basal levels of norepinephrine and ATP were found to be 363nM and 125nM, respectively (n=6). The limit of detection for norepinephrine is 80nM using microchip-based amperometric detection. The LOD for on-line ATP detection using chemiluminescence is 35nM. In previous studies, the co-transmitters have been separated and detected with HPLC techniques. With HPLC, the samples from biological preparations have to be derivatized for ATP detection and require collection time before analysis. Thus real-time measurements are not made and the delay in analysis by HPLC can cause degradation. In conclusion, the method described in the paper can be used to successfully detect norepinephrine and ATP simultaneously and in a near-real-time fashion. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrostatic pressure activates ATP-sensitive K+ channels in lung epithelium by ATP release through pannexin and connexin hemichannels.

PubMed

Richter, Katrin; Kiefer, Kevin P; Grzesik, Benno A; Clauss, Wolfgang G; Fronius, Martin

2014-01-01

Lungs of air-breathing vertebrates are constantly exposed to mechanical forces and therefore are suitable for investigation of mechanotransduction processes in nonexcitable cells and tissues. Freshly dissected Xenopus laevis lungs were used for transepithelial short-circuit current (ISC) recordings and were exposed to increased hydrostatic pressure (HP; 5 cm fluid column, modified Ussing chamber). I(SC) values obtained under HP (I(5cm)) were normalized to values before HP (I(0cm)) application (I(5cm)/I(0cm)). Under control conditions, HP decreased I(SC) (I(5cm)/I(0cm)=0.84; n=68; P<0.0001). This effect was reversible and repeatable ≥30 times. Preincubation with ATP-sensitive K(+) channel (K(ATP)) inhibitors (HMR1098 and glibenclamide) prevented the decrease in I(SC) (I(5cm)/I(0cm): HMR1098=1.19, P<0.0001; glibenclamide=1.11, P<0.0001). Similar effects were observed with hemichannel inhibitors (I(5cm)/I(0cm): meclofenamic acid=1.09, P<0.0001; probenecid=1.0, P<0.0001). The HP effect was accompanied by release of ATP (P<0.05), determined by luciferin-luciferase luminescence in perfusion solution from the luminal side of an Ussing chamber. ATP release was abrogated by both meclofenamic acid and probenecid. RT-PCR experiments revealed the expression of pannexin and connexin hemichannels and KATP subunit transcripts in X. laevis lung. These data show an activation of KATP in pulmonary epithelial cells in response to HP that is induced by ATP release through mechanosensitive pannexin and connexin hemichannels. These findings represent a novel mechanism of mechanotransduction in nonexcitable cells.

Neuronal somatic ATP release triggers neuron–satellite glial cell communication in dorsal root ganglia

PubMed Central

Zhang, X.; Chen, Y.; Wang, C.; Huang, L.-Y. M.

2007-01-01

It has been generally assumed that the cell body (soma) of a neuron, which contains the nucleus, is mainly responsible for synthesis of macromolecules and has a limited role in cell-to-cell communication. Using sniffer patch recordings, we show here that electrical stimulation of dorsal root ganglion (DRG) neurons elicits robust vesicular ATP release from their somata. The rate of release events increases with the frequency of nerve stimulation; external Ca2+ entry is required for the release. FM1–43 photoconversion analysis further reveals that small clear vesicles participate in exocytosis. In addition, the released ATP activates P2X7 receptors in satellite cells that enwrap each DRG neuron and triggers the communication between neuronal somata and glial cells. Blocking L-type Ca2+ channels completely eliminates the neuron–glia communication. We further show that activation of P2X7 receptors can lead to the release of tumor necrosis factor-α (TNFα) from satellite cells. TNFα in turn potentiates the P2X3 receptor-mediated responses and increases the excitability of DRG neurons. This study provides strong evidence that somata of DRG neurons actively release transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. These results also suggest that, contrary to the conventional view, neuronal somata have a significant role in cell–cell signaling. PMID:17525149

Dynamics of shear-induced ATP release from red blood cells.

PubMed

Wan, Jiandi; Ristenpart, William D; Stone, Howard A

2008-10-28

Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release.

The regulation of ATP release from the urothelium by adenosine and transepithelial potential.

PubMed

Dunning-Davies, Bryony M; Fry, Christopher H; Mansour, Dina; Ferguson, Douglas R

2013-03-01

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself. Adenosine, a breakdown product of ATP, is a potent inhibitor of stretch-induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6μM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor-dependent pathway. To test the hypothesis that distension-induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential (TEP). To examine the role of changes to intracellular [Ca(2+) ] in ATP release. Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face. The TEP and short-circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin-luciferase assay. Intracellular [Ca(2+) ] was measured in isolated urothelial cells using the fluorochrome Fura-2. All experiments were performed at 37°C. Distension-induced ATP release was decreased by adenosine (1-10 μm) and enhanced by adenosine deaminase and A1- (but not A2-) receptor antagonists. Distension-induced ATP release was reduced by 2-APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension. ATP and capsaicin, but not adenosine, generated intracellular Ca(2+) transients; adenosine did not affect the ATP-generated Ca(2+) transient. ATP release was dependent on a finite transepithelial potential. Changes to TEP, in the absence of distension, generated ATP release that was in turn reduced by adenosine

Contraction of small mesenteric arteries induced by micromolar concentrations of ATP released from caged ATP.

PubMed

Sjöblom-Widfeldt, N; Arner, A; Nilsson, H

1993-01-01

The concentration dependence of ATP-induced contractions in isolated resistance arteries was estimated using photolysis of caged ATP. Rat mesenteric vessels were isolated and mounted for force registration in a small chamber allowing illumination from a xenon-flash lamp. Photolysis of 100 microM caged ATP, which released about 20 microM ATP within a few milliseconds in the vessel, induced a transient contraction with an amplitude approximately 40-50% of the response induced by 10 microM noradrenaline. The responses could neither be induced by the light flash as such nor by caged ATP alone nor by photolysis of caged phosphate. The amplitude of the contractions was dependent on the concentration of caged ATP, and the effective concentration for ATP was estimated to be in the range of 1-10 microM. In contrast, when ATP was introduced by diffusion, about a 100-fold higher concentration was required. Thus photolytic release of ATP minimizes metabolism before its action on receptors and reveals action of ATP in a concentration range consistent with a role of ATP as a transmitter in nervous regulation of the tone of resistance vessels.

ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL-1beta and IL-18 secretion in an autocrine way.

PubMed

Piccini, Alessandra; Carta, Sonia; Tassi, Sara; Lasiglié, Denise; Fossati, Gianluca; Rubartelli, Anna

2008-06-10

IL-1beta and IL-18 are crucial mediators of inflammation, and a defective control of their release may cause serious diseases. Yet, the mechanisms regulating IL-1beta and IL-18 secretion are partially undefined. Both cytokines are produced as inactive cytoplasmic precursors. Processing to the active form is mediated by caspase-1, which is in turn activated by the multiprotein complex inflammasome. Here, we show that in primary human monocytes microbial components acting on different pathogen-sensing receptors and the danger-associated molecule uric acid are all competent to induce maturation and secretion of IL-1beta and IL-18 through a process that involves as a first event the extracellular release of endogenous ATP. ATP release is followed by autocrine stimulation of the purinergic receptors P2X(7). Indeed, antagonists of the P2X(7) receptor (P2X(7)R), or treatment with apyrase, prevent IL-1beta and IL-18 maturation and secretion triggered by the different stimuli. At variance, blocking P2X(7)R activity has no effects on IL-1beta secretion by monocytes carrying a mutated inflammasome that does not require exogenous ATP for activation. P2X(7)R engagement is followed by K+ efflux and activation of phospholipase A(2). Both events are required for processing and secretion induced by all of the stimuli. Thus, stimuli acting on different pathogen-sensing receptors converge on a common pathway where ATP externalization is the first step in the cascade of events leading to inflammasome activation and IL-1beta and IL-18 secretion.

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

PubMed Central

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

2012-01-01

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

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.

Diadenosine tetraphosphate stimulates atrial ANP release via A(1) receptor: involvement of K(ATP) channel and PKC.

PubMed

Yuan, Kuichang; Cao, Chunhua; Bai, Guang Yi; Kim, Sung Zoo; Kim, Suhn Hee

2007-07-01

Diadenosine polyphosphates (APnAs) are endogenous compounds and exert diverse cardiovascular functions. However, the effects of APnAs on atrial ANP release and contractility have not been studied. In this study, the effects of diadenosine tetraphosphate (AP4A) on atrial ANP release and contractility, and their mechanisms were studied using isolated perfused rat atria. Treatment of atria with AP4A resulted in decreases in atrial contractility and extracellular fluid (ECF) translocation whereas ANP secretion and cAMP levels in perfusate were increased in a dose-dependent manner. These effects of AP4A were attenuated by A(1) receptor antagonist but not by A(2A) or A(3) receptor antagonist. Other purinoceptor antagonists also did not show any effects on AP4A-induced ANF release and contractility. The increment of ANP release and negative inotropy induced by AP4A was similar to those induced by AP3A, AP5A, and AP6A. Protein kinase A inhibitors accentuated AP4A-induced ANP secretion. In contrast, an inhibitor of phospholipase C, protein kinase C or sarcolemma K(ATP) channel completely blocked AP4A-induced ANP secretion. However, an inhibitor of adenylyl cyclase or mitochondria K(ATP) channel had no significant modification of AP4A effects. These results suggest that AP4A regulates atrial inotropy and ANP release mainly through A(1) receptor signaling involving phospholipase C-protein kinase C and sarcolemmal K(ATP) channel and that protein kinase A negatively modulates the effects of AP4A.

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

PubMed Central

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-01-01

HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

PubMed

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-06-23

HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

PubMed

Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

2018-04-10

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity

PubMed Central

Lovatt, Ditte; Xu, Qiwu; Liu, Wei; Takano, Takahiro; Smith, Nathan A.; Schnermann, Jurgen; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca2+ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity. PMID:22421436

Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

PubMed Central

Wall, Mark J; Dale, Nicholas

2013-01-01

The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

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.

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

ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes--a possible sensory mechanism?

PubMed Central

Ferguson, D R; Kennedy, I; Burton, T J

1997-01-01

1. The responses of rabbit urinary bladder to hydrostatic pressure changes and to electrical stimulation have been investigated using both the Ussing chamber and a superfusion apparatus. These experiments enabled us to monitor changes in both ionic transport across the tissue and cellular ATP release from it. 2. The urinary bladder of the rabbit maintains an electrical potential difference across its wall as a result largely of active sodium transport from the urinary (mucosal) to the serosal surface. 3. Small hydrostatic pressure differences produced by removal of bathing fluid from one side of the tissue caused reproducible changes in both potential difference and short-circuit current. The magnitude of these changes increases as the volume of fluid removed increases. 3. Amiloride on the mucosal (urinary), but not the serosal, surface of the membrane reduces the transepithelial potential difference and short-circuit current with an IC50 of 300 nM. Amiloride reduces the size of, but does not abolish, transepithelial potential changes caused by alterations in hydrostatic pressure. 4. Field electrical stimulation of strips of bladder tissue produces a reproducible release of ATP. Such release was demonstrated to occur largely from urothelial cells and is apparently non-vesicular as it increases in the absence of calcium and is not abolished by tetrodotoxin. 5. It is proposed that ATP is released from the urothelium as a sensory mediator for the degree of distension of the rabbit urinary bladder and other sensory modalities. PMID:9423189

Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release.

PubMed

Forsyth, Alison M; Wan, Jiandi; Owrutsky, Philip D; Abkarian, Manouk; Stone, Howard A

2011-07-05

RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold.

Spike-independent release of ATP from Xenopus spinal neurons evoked by activation of glutamate receptors

PubMed Central

Brown, Paul; Dale, Nicholas

2002-01-01

As the release of ATP from neurons has only been directly studied in a few cases, we have used patch sniffing to examine ATP release from Xenopus spinal neurons. ATP release was detected following intracellular current injection to evoke spikes. However, spiking was not essential as both glutamate and NMDA could evoke release of ATP in the presence of TTX. Neither acetylcholine nor high K+ was effective at inducing ATP release in the presence of TTX. Although Cd2+ blocked glutamate-evoked release of ATP suggesting a dependence on Ca2+ entry, neither ω-conotoxin-GVIA nor nifedipine prevented ATP release. N-type and L-type channels are thus not essential for glutamate-evoked ATP release. That glutamate receptors can elicit release in the absence of spiking suggests a close physical relationship between these receptors, the Ca2+ channels and release sites. As the dependence of ATP release on the influx of Ca2+ through Ca2+ channel subtypes differs from that of synaptic transmitter release, ATP may be released from sites that are distinct from those of the principal transmitter. In addition to its role as a fast transmitter, ATP may thus be released as a consequence of the activation of excitatory glutamatergic synapses and act to signal information about activity patterns in the nervous system. PMID:11986374

Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions

PubMed Central

Sui, Guiping; Fry, Chris H.; Montgomery, Bruce; Roberts, Max; Wu, Rui

2013-01-01

The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies. PMID:24285497

Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions.

PubMed

Sui, Guiping; Fry, Chris H; Montgomery, Bruce; Roberts, Max; Wu, Rui; Wu, Changhao

2014-02-01

The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca²⁺ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M₂-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M₂-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca²⁺ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.

Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

PubMed

Davis, Kim A; Samson, Sue E; Wilson, John X; Grover, Ashok K

2006-10-24

In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

PubMed Central

Button, Brian; Picher, Maryse; Boucher, Richard C

2007-01-01

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na+-channel-mediated Na+ absorption and stimulation of Cl− secretion through CFTR and the Ca2+-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence. PMID:17317749

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

PubMed Central

Okada, Seiko F.; Ribeiro, Carla M. P.; Sesma, Juliana I.; Seminario-Vidal, Lucia; Abdullah, Lubna H.; van Heusden, Catharina; Lazarowski, Eduardo R.

2013-01-01

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion. PMID:23763446

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2015-10-15

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus1

PubMed Central

Frenguelli, Bruno G; Wigmore, Geoffrey; Llaudet, Enrique; Dale, Nicholas

2007-01-01

Abstract Adenosine is well known to be released during cerebral metabolic stress and is believed to be neuroprotective. ATP release under similar circumstances has been much less studied. We have now used biosensors to measure and compare in real time the release of ATP and adenosine during in vitro ischaemia in hippocampal slices. ATP release only occurred following the anoxic depolarisation, whereas adenosine release was apparent almost immediately after the onset of ischaemia. ATP release required extracellular Ca2+. By contrast adenosine release was enhanced by removal of extracellular Ca2+, whilst TTX had no effect on either ATP release or adenosine release. Blockade of ionotropic glutamate receptors substantially enhanced ATP release, but had only a modest effect on adenosine release. Carbenoxolone, an inhibitor of gap junction hemichannels, also greatly enhanced ischaemic ATP release, but had little effect on adenosine release. The ecto-ATPase inhibitor ARL 67156, whilst modestly enhancing the ATP signal detected during ischaemia, had no effect on adenosine release. Adenosine release during ischaemia was reduced by pre-treament with homosysteine thiolactone suggesting an intracellular origin. Adenosine transport inhibitors did not inhibit adenosine release, but instead they caused a twofold increase of release. Our data suggest that ATP and adenosine release during ischaemia are for the most part independent processes with distinct underlying mechanisms. These two purines will consequently confer temporally distinct influences on neuronal and glial function in the ischaemic brain. PMID:17459147

Overactive and Underactive Bladder Dysfunction is Reflected by Alterations in Urothelial ATP and NO Release

PubMed Central

Munoz, Alvaro; Smith, Christopher P.; Boone, Timothy B.; Somogyi, George T.

2011-01-01

ATP and NO are released from the urothelium in the bladder. Detrusor Overactivity (DO) following spinal cord injury results in higher ATP and lower NO release from the bladder urothelium. Our aim was to study the relationship between ATP and NO release in 1) early diabetic bladders, an overactive bladder model; and 2) in “diuretic” bladders, an underactive bladder model. To induce diabetes mellitus female rats received 65 mg/kg streptozocin (i.v.). To induce chronic diuresis rats were fed with 5% sucrose. At 28 days, in vivo open cystometry was performed. Bladder wash was collected to analyze the amount of ATP and NO released into the bladder lumen. For in vitro analysis of ATP and NO release, a Ussing chamber was utilized and hypoosmotic Krebs was perfused on the urothelial side of the chamber. ATP was analyzed with luminometry or HPLC-fluorometry while NO was measured with a Sievers NO-analyzer. In vivo ATP release was increased in diabetic bladders and unchanged in diuretic bladders. In vitro release from the urothelium followed the same pattern. NO release was unchanged both in vitro and in vivo in overactive bladders whereas it was enhanced in underactive bladders. We found that the ratio of ATP/NO, representing sensory transmission in the bladder, was high in overactive and low in underactive bladder dysfunction. In summary, ATP release has a positive correlation while NO release has a negative correlation with the bladder contraction frequency. The urinary ATP/NO ratio may be a clinically relevant biomarker to characterize the extent of bladder dysfunction. PMID:21145365

The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

NASA Astrophysics Data System (ADS)

Forsyth, Alison; Abkarian, Manouk; Wan, Jiandi; Stone, Howard

2010-11-01

In shear flow, red blood cells (RBCs) exhibit a variety of behaviors such as rouleaux formation, tumbling, swinging, and tank-treading. The physiological consequences of these dynamic behaviors are not understood. In vivo, ATP is known to signal vasodilation; however, to our knowledge, no one has deciphered the relevance of RBC microrheology to the functional release of ATP. Previously, we correlated RBC deformation and ATP release in microfluidic constrictions (Wan et al., 2008). In this work, a cone-plate rheometer is used to shear a low hematocrit solution of RBCs at varying viscosity ratios (λ) between the inner cytoplasmic hemoglobin and the outer medium, to determine the intrinsic viscosity of the suspension. Further, using a luciferin-luciferase enzymatic reaction, we report the relative ATP release at varying shear rates. Results indicate that for λ = 1.6, 3.8 and 11.1, ATP release is constant up to 500 s-1, which suggests that the tumbling-tanktreading transition does not alter ATP release in pure shear. For lower viscosity ratios, λ = 1.6 and 3.8, at 500 s-1 a change in slope occurs in the intrinsic viscosity data and is marked by an increase in ATP release. Based on microfluidic observations, this simultaneous change in viscosity and ATP release occurs within the tank-treading regime.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter*

PubMed Central

Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

2015-01-01

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [3H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. PMID:25991724

P2X7 receptor-stimulation causes fever via PGE2 and IL-1β release.

PubMed

Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Gomez, Ana I; Machado, Francisco; Di Virgilio, Francesco; Pelegrín, Pablo

2012-07-01

Prostaglandins (PGs) are important lipid mediators involved in the development of inflammatory associated pain and fever. PGE2 is a well-established endogenous pyrogen activated by proinflammatory cytokine interleukin (IL)-1β. P2X7 receptors (P2X7Rs) expressed by inflammatory cells are stimulated by the danger signal extracellular ATP to activate the inflammasome and release IL-1β. Here we show that P2X7R activation is required for the release of PGE2 and other autacoids independent of inflammasome activation, with an ATP EC(50) for PGE2 and IL-1β release of 1.58 and 1.23 mM, respectively. Furthermore, lack of P2X7R or specific antagonism of P2X7R decreased the febrile response in mice triggered after intraperitoneal LPS or IL-1β inoculation. Accordingly, LPS inoculation caused intraperitoneal ATP accumulation. Therefore, P2X7R antagonists emerge as novel therapeutics for the treatment for acute inflammation, pain and fever, with wider anti-inflammatory activity than currently used cyclooxygenase inhibitors.-Barberà-Cremades, M., Baroja-Mazo, A., Gomez, A. I., Machado, F., Di Virgilio, F., Pelegrín, P. P2X7 receptor-stimulation causes fever via PGE2 and IL-1β release.

Cell stress increases ATP release in NLRP3 inflammasome-mediated autoinflammatory diseases, resulting in cytokine imbalance

PubMed Central

Carta, Sonia; Penco, Federica; Lavieri, Rosa; Martini, Alberto; Dinarello, Charles Anthony; Gattorno, Marco; Rubartelli, Anna

2015-01-01

Cell stress is implicated in triggering bouts of systemic inflammation in patients with autoinflammatory disorders. Blood monocytes from patients affected by NLRP3-mediated cryopyrin-associated periodic syndromes (CAPS) release greater amounts of IL-1β than monocytes from unaffected subjects. Here we show that stress lowers the threshold of activation; blood monocytes from CAPS patients maintain the high levels of secreted IL-1β (fivefold) and IL-18 (10-fold) when stimulated with 1,000-fold less LPS than that required for full IL-1β secretion in control subjects. Unexpectedly, IL-1α secretion is increased 10-fold, indicating that inflammatory episodes in CAPS may not be entirely a result of IL-1β but may also involve IL-1α. In CAPS monocytes, LPS induces the externalization of copious amounts of ATP (10-fold), which drive IL-1β, IL-18, and IL-1α release via activation of the P2X purinoceptor 7. This enhanced ATP release appears to be the link between cell stress and increased cytokine secretion in CAPS. In the later phase after LPS stimulation, CAPS monocytes undergo oxidative stress, which impairs production of the anti-inflammatory IL-1 receptor antagonist (IL-1Ra). Remarkably, IL-1Ra secretion is fully restored by treatment with antioxidants. In two patients with the same NLRP3 mutation, but different disease severity, monocytes from the mildly affected patient exhibited more efficient redox response, lower ATP secretion, and more balanced cytokine production. Thus, the robustness of the individual antioxidant response increases the tolerance to stress and reduces the negative effect of the disease. Pharmacologic block of P2X purinoceptor 7 and improved stress tolerance may represent novel treatment strategies in stress-associated inflammatory diseases. PMID:25730877

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

PubMed

Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

2015-07-03

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A taste for ATP: neurotransmission in taste buds

PubMed Central

Kinnamon, Sue C.; Finger, Thomas E.

2013-01-01

Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

PubMed

Sabirov, R Z; Dutta, A K; Okada, Y

2001-09-01

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

ATP hydrolysis assists phosphate release and promotes reaction ordering in F1-ATPase

PubMed Central

Li, Chun-Biu; Ueno, Hiroshi; Watanabe, Rikiya; Noji, Hiroyuki; Komatsuzaki, Tamiki

2015-01-01

F1-ATPase (F1) is a rotary motor protein that can efficiently convert chemical energy to mechanical work of rotation via fine coordination of its conformational motions and reaction sequences. Compared with reactant binding and product release, the ATP hydrolysis has relatively little contributions to the torque and chemical energy generation. To scrutinize possible roles of ATP hydrolysis, we investigate the detailed statistics of the catalytic dwells from high-speed single wild-type F1 observations. Here we report a small rotation during the catalytic dwell triggered by the ATP hydrolysis that is indiscernible in previous studies. Moreover, we find in freely rotating F1 that ATP hydrolysis is followed by the release of inorganic phosphate with low synthesis rates. Finally, we propose functional roles of the ATP hydrolysis as a key to kinetically unlock the subsequent phosphate release and promote the correct reaction ordering. PMID:26678797

ATP release from bladder urothelium and serosa in a rat model of partial bladder outlet obstruction.

PubMed

Shiina, Kazuhiro; Hayashida, Ken-Ichiro; Ishikawa, Kazuo; Kawatani, Masahito

2016-01-01

Overactive bladder is one of the major health problem especially in elderly people. Adenosine triphosphate (ATP) is released from urinary bladder cells and acts as a smooth muscle contraction and sensory signal in micturition but little is known about the role of ATP release in the pathophysiology of overactive bladder. To assess the relationship between ATP and overactive bladder, we used a partial bladder outlet obstruction (pBOO) model in rats. The bladder caused several changes by pBOO: An increase in bladder weight, hypertrophy of sub-urothelium and sub-serosal area, and frequent non-voiding bladder contraction during urine storage. Basal ATP release from urothelium and serosa of pBOO rats was significantly higher than that of normal rats. Distentioninduced ATP release from urothelium of normal and pBOO rats had no significant change. However, distention-induced ATP release from serosa of pBOO rats was higher than that of normal. These findings may identify ATP especially released from serosa as one of causes of non-voiding contractions and overactive bladder symptoms.

ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

PubMed Central

Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

2011-01-01

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

Calcium released by photolysis of DM-nitrophen stimulates transmitter release at squid giant synapse.

PubMed

Delaney, K R; Zucker, R S

1990-07-01

1. Transmitter release at the squid giant synapse was stimulated by photolytic release of Ca2+ from the 'caged' Ca2+ compound DM-nitrophen (Kaplan & Ellis-Davies, 1988) inserted into presynaptic terminals. 2. Competing binding reactions cause the amount of Ca2+ released by DM-nitrophen photolysis to depend on the concentrations of DM-nitrophen, total Ca2+, Mg+, ATP and native cytoplasmic Ca2+ buffer. Measurements of presynaptic [Ca2+] changes by co-injection of the fluorescent indicator dye Fura-2 show that DM-nitrophen photolysis causes a transient rise in Ca2+ followed by decay within about 150 ms to an increased steady-state level. 3. Rapid photolysis of Ca2(+)-loaded nitrophen within the presynaptic terminal was followed in less than a millisecond by depolarization of the postsynaptic membrane. As with action potential-evoked excitatory postsynaptic potentials (EPSPs), the light-evoked response was partially and reversibly blocked by 1-3 mM-kainic acid which desensitizes postsynaptic glutamate receptors. 4. Release was similar in magnitude and rate to normal action potential-mediated EPSPs. 5. The release of transmitter by photolysis of Ca2(+)-loaded DM-nitrophen was not affected by removal of Ca2+ from the saline or addition of tetrodotoxin. Photolysis of DM-nitrophen injected into presynaptic terminals without added Ca2+ did not stimulate release of transmitter nor did it interfere with normal action potential-mediated release. 6. Stimulation of presynaptic action potentials in Ca2(+)-free saline during the light-evoked response did not elicit increased release of transmitter if the ganglion was bathed in Ca2(+)-free saline, i.e. in the absence of Ca2+ influx. Increasing the intensity of the light or stimulating presynaptic action potentials in Ca2(+)-containing saline increased the release of transmitter. Therefore the failure of presynaptic voltage change to increase transmitter release resulting from release of caged Ca2+ was not due to saturation or

Role of connexin43 hemichannels in mechanical stress-induced ATP release in human periodontal ligament cells.

PubMed

Luckprom, P; Kanjanamekanant, K; Pavasant, P

2011-10-01

Our previous studies showed that mechanical stress could induce ATP release in human periodontal ligament (HPDL) cells. By signaling through P2 purinergic receptors, ATP increased the expression and the synthesis of osteopontin and RANKL. In this study, the mechanism of stress-induced ATP release was investigated. Continuous compressive forces were applied on cultured HPDL cells. The ATP released was measured using luciferin-luciferase bioluminescence. The expression of gap-junction proteins was examined using RT-PCR and western blot analysis. The opening of hemichannels was demonstrated by cellular uptake of a fluorescent dye, 5(6)-carboxyfluorescein, which is known to penetrate hemichannels. Intracellular signal transduction was investigated using inhibitors and antagonists. Mechanical stress induced the release of ATP into the culture medium, which was attenuated by carbenoxolone, a nonspecific gap-junction inhibitor. Addition of meclofenamic acid sodium salt, a connexin43 inhibitor, inhibited ATP release by mechanical stress. Knockdown of connexin43 expression by small interfering RNA reduced the amount of ATP released by mechanical stress, suggesting the role of connexin43 hemichannels. In addition, intracellular Ca(2+) blockers could also inhibit mechanical stress-induced ATP release and the opening of the gap junction. Our study demonstrated the involvement of gap-junction hemichannels, especially connexin43, in the stress-induced ATP-release mechanism. Furthermore, this mechanism may be regulated by the intracellular Ca(2+) signaling pathway. These results suggest an important role of gap-junction hemichannels in the function and behavior of HPDL cells. © 2011 John Wiley & Sons A/S.

The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release.

PubMed

Forsyth, Alison M; Braunmüller, Susanne; Wan, Jiandi; Franke, Thomas; Stone, Howard A

2012-05-01

It is known that deformation of red blood cells (RBCs) is linked to ATP release from the cells. Further, membrane cholesterol has been shown to alter properties of the cell membrane such as fluidity and bending stiffness. Membrane cholesterol content is increased in some cardiovascular diseases, for example, in individuals with acute coronary syndromes and chronic stable angina, and therefore, because of the potential clinical relevance, we investigated the influence of altered RBC membrane cholesterol levels on ATP release. Because of the correlation between statins and reduced membrane cholesterol in vivo, we also investigated the effects of simvastatin on RBC deformation and ATP release. We found that reducing membrane cholesterol increases cell deformability and ATP release. We also found that simvastatin increases deformability by acting directly on the membrane in the absence of the liver, and that ATP release was increased for cells with enriched cholesterol after treatment with simvastatin. Copyright © 2012 Elsevier Inc. All rights reserved.

ATP during early bladder stretch is important for urgency in detrusor overactivity patients.

PubMed

Cheng, Y; Mansfield, K J; Allen, W; Chess-Williams, R; Burcher, E; Moore, K H

2014-01-01

ATP is an important mediator of urgency in women with detrusor overactivity (DO). In order to understand how different degrees of bladder stretch elicited ATP release in DO patients compared with controls, sequential aliquots were collected during cystometry and ATP release was measured at each degree of bladder filling, in female patients with DO and controls. In both DO and control groups, ATP release was induced during bladder filling, suggesting that stretch stimulated further ATP release. However, the luminal ATP concentrations were already high at early filling stage (200 mL), which was even greater than those at the later filling stages (400 mL and maximum cystometric capacity, MCC), indicating that a substantial ATP release has been induced during early filling (200 mL) in both DO and controls. In DO, ATP release at 200 mL was significantly higher in those with low first desire to void (FDV) (≤ 200 mL) than in those with higher FDV (> 200 mL); this may suggest that ATP release at early stretch may play an important role in urgency (early sensation) in DO. ATP concentrations remained unchanged after voiding, suggesting that voiding did not further induce ATP release into intraluminal fluid.

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

PubMed

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Ca2+-mediated ascorbate release from coronary artery endothelial cells.

PubMed

Davis, Kim A; Samson, Sue E; Best, Kelly; Mallhi, Kanwaldeep K; Szewczyk, Magdalena; Wilson, John X; Kwan, Chiu-Yin; Grover, Ashok K

2006-01-01

1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C.

PubMed

Weng, Ju-Yun; Hsu, Tsan-Ting; Sun, Synthia H

2008-05-15

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes. Copyright 2007 Wiley-Liss, Inc.

Impaired ATP release from red blood cells promotes their adhesion to endothelial cells: A mechanism of hypoxemia after transfusion

PubMed Central

Zhu, Hongmei; Zennadi, Rahima; Xu, Bruce X.; Eu, Jerry P.; Torok, Jordan A.; Telen, Marilyn J.; McMahon, Timothy J.

2011-01-01

Objective Transfusion of red blood cells (RBCs) has been linked to disappointing clinical outcomes in the critically ill, but specific mechanisms of organ dysfunction after transfusion remain poorly understood. We tested the hypothesis that RBC storage impairs the ability of RBCs to release ATP and that impaired ATP-release was injurious in vivo, in part through increased RBC adhesion. Design Prospective, controlled, mechanistic study. Setting University research laboratory. Subjects Human and mouse blood donors; nude mouse transfusion recipients. Interventions Manipulation of ATP release, supplemental ATP, and antibodies to RBC and endothelial adhesion receptors were used in vitro and in vivo to probe the roles of released ATP and adhesion in responses to (transfused) RBCs. Measurements and main results The ability of stored RBCs to release ATP declined markedly within 14 days after collection, despite relatively stable levels of ATP within the RBCs. Inhibiting ATP release promoted the adhesion of stored RBCs to endothelial cells in vitro and RBC sequestration in the lungs of transfused mice in vivo. Unlike transfusion of fresh human RBCs, stored-RBC transfusion in mice decreased blood oxygenation and increased extravasation of RBCs into the lung’s alveolar airspaces. Similar findings were seen with transfusion of fresh RBCs treated with the ATP-release inhibitors glibenclamide and carbenoxolone. These findings were prevented by either co-infusion of an ATP analog or pre-transfusion incubation of the RBCs with an antibody against the erythrocyte adhesion receptor LW (Landsteiner-Wiener; ICAM-4). Conclusions The normal flow of RBCs in pulmonary microvessels depends in part on the release of anti-adhesive ATP from RBCs, and storage-induced deficiency in ATP release from transfused RBCs may promote or exacerbate microvascular pathophysiology in the lung, in part through increased RBC adhesion. PMID:21765360

Alpha-ketoisocaproate is not a true substrate for ATP production by pancreatic beta-cell mitochondria.

PubMed

Lembert, N; Idahl, L A

1998-03-01

The ability of alpha-ketoisocaproate (KIC) to induce ATP production in isolated mitochondria from pancreatic beta-cells was examined with a bioluminometric method. There was no ATP production from KIC when tested alone or in combination with malate (1 mmol/l), nor did DL-beta-hydroxybutyrate induce mitochondrial ATP production, whereas palmitoyl-carnitine and pyruvate were efficient stimulators of mitochondrial ATP production in the presence of an equimolar concentration of malate. However, KIC stimulated the mitochondrial ATP production when tested in combination with glutamate (10 mmol/l). The concentration necessary to obtain half-maximal stimulation was approximately 50 micromol/l KIC, and maximal activity, comparable to that obtained with fatty acids, was reached at 1 mmol/l KIC. Higher KIC concentrations inhibited the mitochondrial ATP production, whereas a plateau was attained at 1 mmol/l KIC in the presence of glutamine. Ca2+ stimulated the maximal mitochondrial ATP production induced by KIC. Maximal stimulation was obtained with 300 nmol/l Ca2+ in the presence of 0.3 mmol/l KIC. Ca2+ reduced the concentration of KIC necessary for half-maximal stimulation to <30 micromol/l. Leucine stimulated the mitochondrial ATP production in the presence of glutamate to the same extent as KIC. Half-maximal stimulation was observed with 2 mmol/l leucine. There were no additive effects on mitochondrial ATP production when KIC and leucine were tested in combination. The results demonstrate that KIC by itself is not a mitochondrial substrate for ATP production. KIC must transaminate with glutamate or glutamine to yield alpha-ketoglutarate and leucine. Since leucine allosterically activates glutamate dehydrogenase, which also produces alpha-ketoglutarate, the insulinogenic effect of KIC may in part be due to the intramitochondrial generation of alpha-ketoglutarate. Since KIC-induced ATP production reaches a plateau already at micromolar concentrations (i.e., far below the

S-Sulfhydration of ATP synthase by hydrogen sulfide stimulates mitochondrial bioenergetics.

PubMed

Módis, Katalin; Ju, YoungJun; Ahmad, Akbar; Untereiner, Ashley A; Altaany, Zaid; Wu, Lingyun; Szabo, Csaba; Wang, Rui

2016-11-01

Mammalian cells can utilize hydrogen sulfide (H 2 S) to support mitochondrial respiration. The aim of our study was to explore the potential role of S-sulfhydration (a H 2 S-induced posttranslational modification, also known as S-persulfidation) of the mitochondrial inner membrane protein ATP synthase (F1F0 ATP synthase/Complex V) in the regulation of mitochondrial bioenergetics. Using a biotin switch assay, we have detected S-sulfhydration of the α subunit (ATP5A1) of ATP synthase in response to exposure to H 2 S in vitro. The H 2 S generator compound NaHS induced S-sulfhydration of ATP5A1 in HepG2 and HEK293 cell lysates in a concentration-dependent manner (50-300μM). The activity of immunocaptured mitochondrial ATP synthase enzyme isolated from HepG2 and HEK293 cells was stimulated by NaHS at low concentrations (10-100nM). Site-directed mutagenesis of ATP5A1 in HEK293 cells demonstrated that cysteine residues at positions 244 and 294 are subject to S-sulfhydration. The double mutant ATP synthase protein (C244S/C294S) showed a significantly reduced enzyme activity compared to control and the single-cysteine-mutated recombinant proteins (C244S or C294S). To determine whether endogenous H 2 S plays a role in the basal S-sulfhydration of ATP synthase in vivo, we compared liver tissues harvested from wild-type mice and mice deficient in cystathionine-gamma-lyase (CSE, one of the three principal mammalian H 2 S-producing enzymes). Significantly reduced S-sulfhydration of ATP5A1 was observed in liver homogenates of CSE -/- mice, compared to wild-type mice, suggesting a physiological role for CSE-derived endogenous H 2 S production in the S-sulfhydration of ATP synthase. Various forms of critical illness (including burn injury) upregulate H 2 S-producing enzymes and stimulate H 2 S biosynthesis. In liver tissues collected from mice subjected to burn injury, we detected an increased S-sulfhydration of ATP5A1 at the early time points post-burn. At later time points

Modulation of Central Synapses by Astrocyte-Released ATP and Postsynaptic P2X Receptors

PubMed Central

Pankratov, Yuriy

2017-01-01

Communication between neuronal and glial cells is important for neural plasticity. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons and/or glia. Recent data show that postsynaptic P2X receptors underlie slow neuromodulatory actions rather than fast synaptic transmission at brain synapses. Here, we review these findings with a particular focus on the release of ATP by astrocytes and the diversity of postsynaptic P2X-mediated modulation of synaptic strength and plasticity in the CNS. PMID:28845311

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

PubMed Central

Ceriani, Federico; Pozzan, Tullio; Mammano, Fabio

2016-01-01

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

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.

Breast feeding increases vasoconstriction induced by electrical field stimulation in rat mesenteric artery. Role of neuronal nitric oxide and ATP.

PubMed

Blanco-Rivero, Javier; Sastre, Esther; Caracuel, Laura; Granado, Miriam; Balfagón, Gloria

2013-01-01

The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications. Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O(2)(.-)), NA and ATP releases were also determined. EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O(2)(.-) production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals. Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats.

Breast Feeding Increases Vasoconstriction Induced by Electrical Field Stimulation in Rat Mesenteric Artery. Role of Neuronal Nitric Oxide and ATP

PubMed Central

Caracuel, Laura; Granado, Miriam; Balfagón, Gloria

2013-01-01

Objectives The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications. Methods Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O2 .–), NA and ATP releases were also determined. Results EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 µmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O2 .– production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals. Conclusion Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats. PMID:23342008

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

Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response.

PubMed

Calder, Bennett W; Matthew Rhett, Joshua; Bainbridge, Heather; Fann, Stephen A; Gourdie, Robert G; Yost, Michael J

2015-06-01

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. 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. 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. These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital

CFTR mediates noradrenaline-induced ATP efflux from DRG neurons.

PubMed

Kanno, Takeshi; Nishizaki, Tomoyuki

2011-09-24

In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β(3) adrenoceptors linked to G(s) protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.

Calcium modulates the ATP and ADP hydrolysis in human placental mitochondria.

PubMed

Martínez, Federico; Uribe, Aida; Espinosa-García, M Teresa; Flores-Herrera, Oscar; García-Pérez, Cecilia; Milán, Rebeca

2002-08-01

This study evaluated the effect of Ca2+ on the extramitochondrial hydrolysis of ATP and ADP by the extramitochondrial ATPase in isolated mitochondria and submitochondrial particles (SMPs) from human term placenta. The effect of different oxidizable substrates on the hydrolysis of ATP and ADP in the presence of sucrose or K+ was evaluated. Ca2+ increased phosphate release from ATP and ADP, but this stimulation showed different behavior depending on the oxidizable substrate present in the incubation media. Ca2+ stimulated the hydrolysis of ATP and ADP in the presence of sucrose. However, Ca2+ did not stimulate the hydrolysis of ADP in the medium containing K+. Ca2+ showed inhibition depending on the respiratory substrate. This study suggests that the energetic state of mitochondria controls the extramitochondrial ATPase activity, which is modulated by Ca2+ and respiratory substrates.

ATP mediates flow-induced NO production in thick ascending limbs

PubMed Central

Hong, Nancy J.; Garvin, Jeffrey L.

2012-01-01

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane. PMID:22496412

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Naohiko; Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp; Furuya, Kishio

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellularmore » Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.« less

Mucosal adenosine triphosphate mediates serotonin release from ileal but not colonic guinea pig enterochromaffin cells.

PubMed

Patel, B A

2014-02-01

Mechanical stimulation of the mucosal epithelium results in increased serotonin (5-HT) release from enterochromaffin (EC) cells. Little is known about how this process varies in different regions of the intestinal tract; however, purines are felt to play a role. We studied the relationship between mechanical stimulation, adenosine triphosphate (ATP), and 5-HT release from ileal and colonic mucosal tissue. Amperometric recordings of ATP and 5-HT were carried out using an ATP biosensor and boron-doped diamond microelectrode. Levels of extracellular ATP and 5-HT were monitored using high performance liquid chromatography. Under basal conditions, 5-HT levels were significantly decreased in the ileum (p < 0.001) but not the colon in the presence of the P2 antagonist suramin (100 μM). Ecto-ATPase inhibitor ARL67156 (10 μM) elevated ATP levels in the ileum and colon (both p < 0.001), but only 5-HT levels in the ileum (p < 0.001). Exogenous ATP increased 5-HT release in the presence of tetrodotoxin in the ileum (p < 0.001), but had not effect in the colon. Mechanical stimulation increased levels of 5-HT in the ileum (p < 0.001) and colon (p < 0.01), but levels returned to baseline in the presence of suramin and MRS2179 in the ileum. The onset of 5-HT release was delayed following mechanical stimulation. The rise time of the ATP response was quicker than that of 5-HT during mechanical stimulation. During mechanical stimulation of the mucosal epithelium, ATP mediates 5-HT release from EC cells in the ileum, but not the colon. Mucosal 5-HT signaling following mechanical stimulation is varied in different regions of the intestinal tract. © 2013 John Wiley & Sons Ltd.

The ATP hydrolysis and phosphate release steps control the time course of force development in rabbit skeletal muscle.

PubMed

Sleep, John; Irving, Malcolm; Burton, Kevin

2005-03-15

The time course of isometric force development following photolytic release of ATP in the presence of Ca(2+) was characterized in single skinned fibres from rabbit psoas muscle. Pre-photolysis force was minimized using apyrase to remove contaminating ATP and ADP. After the initial force rise induced by ATP release, a rapid shortening ramp terminated by a step stretch to the original length was imposed, and the time course of the subsequent force redevelopment was again characterized. Force development after ATP release was accurately described by a lag phase followed by one or two exponential components. At 20 degrees C, the lag was 5.6 +/- 0.4 ms (s.e.m., n = 11), and the force rise was well fitted by a single exponential with rate constant 71 +/- 4 s(-1). Force redevelopment after shortening-restretch began from about half the plateau force level, and its single-exponential rate constant was 68 +/- 3 s(-1), very similar to that following ATP release. When fibres were activated by the addition of Ca(2+) in ATP-containing solution, force developed more slowly, and the rate constant for force redevelopment following shortening-restretch reached a maximum value of 38 +/- 4 s(-1) (n = 6) after about 6 s of activation. This lower value may be associated with progressive sarcomere disorder at elevated temperature. Force development following ATP release was much slower at 5 degrees C than at 20 degrees C. The rate constant of a single-exponential fit to the force rise was 4.3 +/- 0.4 s(-1) (n = 22), and this was again similar to that after shortening-restretch in the same activation at this temperature, 3.8 +/- 0.2 s(-1). We conclude that force development after ATP release and shortening-restretch are controlled by the same steps in the actin-myosin ATPase cycle. The present results and much previous work on mechanical-chemical coupling in muscle can be explained by a kinetic scheme in which force is generated by a rapid conformational change bracketed by two

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

PubMed

Bele, Tanja; Fabbretti, Elsa

2016-08-01

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.

PubMed

Dautova, Yana; Kapustin, Alexander N; Pappert, Kevin; Epple, Matthias; Okkenhaug, Hanneke; Cook, Simon J; Shanahan, Catherine M; Bootman, Martin D; Proudfoot, Diane

2018-02-01

Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. Using ELISA to measure IL-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1β release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1β release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1β release. CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

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.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium.

PubMed

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-07-01

Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [(3) H]-ACh overflow experiments. Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [(3) H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. © 2014 The British Pharmacological Society.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

PubMed Central

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-01-01

BACKGROUND AND PURPOSE Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. EXPERIMENTAL APPROACH We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [3H]-ACh overflow experiments. KEY RESULTS Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [3H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. CONCLUSIONS AND IMPLICATIONS Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. PMID:24697602

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

PubMed

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

2010-04-01

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

DNA Stimulates ATP-Dependent Proteolysis and Protein-Dependent ATPase Activity of Protease La from Escherichia coli

NASA Astrophysics Data System (ADS)

Chung, Chin Ha; Goldberg, Alfred L.

1982-02-01

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhanced ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.

Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

PubMed

Matos, R; Cordeiro, J M; Coelho, A; Ferreira, S; Silva, C; Igawa, Y; Cruz, F; Charrua, A

2016-12-01

Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Release of ATP from marginal cells in the cochlea of neonatal rats can be induced by changes in extracellular and intracellular ion concentrations.

PubMed

Peng, Yating; Chen, Jie; He, Shan; Yang, Jun; Wu, Hao

2012-01-01

Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Sprague-Dawley rats aged 1-3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K(+) and intra- and extracellular Ca(2+). Furthermore, changes in the concentration of intracellular Ca(2+) induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K(+) channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A(2).

Release of ATP from Marginal Cells in the Cochlea of Neonatal Rats Can Be Induced by Changes in Extracellular and Intracellular Ion Concentrations

PubMed Central

Peng, Yating; Chen, Jie; He, Shan; Yang, Jun; Wu, Hao

2012-01-01

Background Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Methods Sprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Results Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. Conclusion We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2. PMID:23071731

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

PubMed Central

Conley, Jason M.

2017-01-01

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens. PMID:29121644

Visualizing dopamine released from living cells using a nanoplasmonic probe

NASA Astrophysics Data System (ADS)

Qin, W. W.; Wang, S. P.; Li, J.; Peng, T. H.; Xu, Y.; Wang, K.; Shi, J. Y.; Fan, C. H.; Li, D.

2015-09-01

We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC).We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC). Electronic supplementary information (ESI) available: Fig. S1-S4 and Table S1. See DOI: 10.1039/c5nr04433b

Lubiprostone stimulates small intestinal mucin release

PubMed Central

2012-01-01

Background Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 (PGE1) used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid secretion. In a mouse model of the genetic disease cystic fibrosis, we previously showed that in vivo administration of lubiprostone resulted in greater mucus accumulation in the small intestine. The aim of this study was to directly test whether lubiprostone stimulates intestinal mucin release. Methods Mucin release was measured by mounting segments (4-5 cm) of mouse proximal-mid small intestine in an organ bath, allowing access to the perfusate (luminal) and the bath (serosal) solutions. Nifedipine (10-6 M) and indomethacin (10-5 M) were included in all solutions to inhibit smooth muscle activity and endogenous prostaglandin production, respectively. The tissue was equilibrated under flow for 30 min, using the perfusate collected during the final 10 min of the equilibration period to measure unstimulated release rate. Stimulus was then added to either the perfusate or the bath and the perfusate was collected for another 30 min to measure the stimulated mucin release rate. Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using purified pig gastric mucin as a standard. Results When applied luminally at 1 μM lubiprostone was ineffective at stimulating mucin release. When added to the serosal solution, 1 μM lubiprostone stimulated mucin release to ~300% of the unstimulated rate. As a positive control, serosal 1 μM prostaglandin E2 increased mucin release to ~400% of the unstimulated rate. Conclusions These results support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. Stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis. Thus, in addition to chronic

Lubiprostone stimulates small intestinal mucin release.

PubMed

De Lisle, Robert C

2012-11-06

Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 (PGE1) used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid secretion. In a mouse model of the genetic disease cystic fibrosis, we previously showed that in vivo administration of lubiprostone resulted in greater mucus accumulation in the small intestine. The aim of this study was to directly test whether lubiprostone stimulates intestinal mucin release. Mucin release was measured by mounting segments (4-5 cm) of mouse proximal-mid small intestine in an organ bath, allowing access to the perfusate (luminal) and the bath (serosal) solutions. Nifedipine (10-6 M) and indomethacin (10-5 M) were included in all solutions to inhibit smooth muscle activity and endogenous prostaglandin production, respectively. The tissue was equilibrated under flow for 30 min, using the perfusate collected during the final 10 min of the equilibration period to measure unstimulated release rate. Stimulus was then added to either the perfusate or the bath and the perfusate was collected for another 30 min to measure the stimulated mucin release rate. Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using purified pig gastric mucin as a standard. When applied luminally at 1 μM lubiprostone was ineffective at stimulating mucin release. When added to the serosal solution, 1 μM lubiprostone stimulated mucin release to ~300% of the unstimulated rate. As a positive control, serosal 1 μM prostaglandin E2 increased mucin release to ~400% of the unstimulated rate. These results support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. Stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis. Thus, in addition to chronic constipation, there is greater potential for the

Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2017-07-01

Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion.

PubMed

Sone, Hideyuki; Sasaki, Yuka; Komai, Michio; Toyomizu, Masaaki; Kagawa, Yasuo; Furukawa, Yuji

2004-02-13

Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.

A highly active ATP-insensitive K+ import pathway in plant mitochondria.

PubMed

Ruy, Fernando; Vercesi, Anibal E; Andrade, Paula B M; Bianconi, M Lucia; Chaimovich, Hernan; Kowaltowski, Alicia J

2004-04-01

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

PubMed Central

Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

PubMed Central

Rasooli-Nejad, Seyed; Andrew, Jemma; Haydon, Philip G.; Pankratov, Yuriy

2014-01-01

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca2+-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca2+-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca2+ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca2+ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca2+-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes

Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

PubMed

Price, Alexander K; Fischer, David J; Martin, R Scott; Spence, Dana M

2004-08-15

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

PubMed Central

Chiesa, Scott T.; Trangmar, Steven J.; Ali, Leena; Lotlikar, Makrand D.; González‐Alonso, José

2017-01-01

New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In

Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Fen; Hui, Zhenhai; Wei, Wei

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca{sup 2+}]{sub c}) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects weremore » almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca{sup 2+} channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca{sup 2+} entry through activation of TRPV4 channel in synoviocytes. - Highlights: • Hypotonic stress evokes Ca{sup 2+} entry in rheumatoid arthritis synovial fibroblasts. • Hypotonic stress induces rapid ATP release and ROS production in synoviocytes. • Hypotonic stimulation promotes the proliferation of synovial fibroblasts. • TRPV4 controls hypotonic-induced responses in synoviocytes.« less

OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium.

PubMed

Collins, Valerie M; Daly, Donna M; Liaskos, Marina; McKay, Neil G; Sellers, Donna; Chapple, Christopher; Grundy, David

2013-11-01

To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium. Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension. Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin-luciferase and Amplex(®) Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively. OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application. Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures. Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing. After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated. OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release. These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves. © 2013 The Authors. BJU International © 2013 BJU International.

Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenhagen, Jason Alan

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 activatedmore » a G{sub q}-type protein to initiate ATP release from HUVECs. Ca 2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca 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 + 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

Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction.

PubMed

Shibukawa, Yoshiyuki; Sato, Masaki; Kimura, Maki; Sobhan, Ubaidus; Shimada, Miyuki; Nishiyama, Akihiro; Kawaguchi, Aya; Soya, Manabu; Kuroda, Hidetaka; Katakura, Akira; Ichinohe, Tatsuya; Tazaki, Masakazu

2015-04-01

Various stimuli induce pain when applied to the surface of exposed dentin. However, the mechanisms underlying dentinal pain remain unclear. We investigated intercellular signal transduction between odontoblasts and trigeminal ganglion (TG) neurons following direct mechanical stimulation of odontoblasts. Mechanical stimulation of single odontoblasts increased the intracellular free calcium concentration ([Ca(2+)]i) by activating the mechanosensitive-transient receptor potential (TRP) channels TRPV1, TRPV2, TRPV4, and TRPA1, but not TRPM8 channels. In cocultures of odontoblasts and TG neurons, increases in [Ca(2+)]i were observed not only in mechanically stimulated odontoblasts, but also in neighboring odontoblasts and TG neurons. These increases in [Ca(2+)]i were abolished in the absence of extracellular Ca(2+) and in the presence of mechanosensitive TRP channel antagonists. A pannexin-1 (ATP-permeable channel) inhibitor and ATP-degrading enzyme abolished the increases in [Ca(2+)]i in neighboring odontoblasts and TG neurons, but not in the stimulated odontoblasts. G-protein-coupled P2Y nucleotide receptor antagonists also inhibited the increases in [Ca(2+)]i. An ionotropic ATP (P2X3) receptor antagonist inhibited the increase in [Ca(2+)]i in neighboring TG neurons, but not in stimulated or neighboring odontoblasts. During mechanical stimulation of single odontoblasts, a connexin-43 blocker did not have any effects on the [Ca(2+)]i responses observed in any of the cells. These results indicate that ATP, released from mechanically stimulated odontoblasts via pannexin-1 in response to TRP channel activation, transmits a signal to P2X3 receptors on TG neurons. We suggest that odontoblasts are sensory receptor cells and that ATP released from odontoblasts functions as a neurotransmitter in the sensory transduction sequence for dentinal pain.

Expression and Roles of Pannexins in ATP Release in the Pituitary Gland

PubMed Central

Li, Shuo; Bjelobaba, Ivana; Yan, Zonghe; Kucka, Marek; Tomić, Melanija

2011-01-01

Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland. PMID:21467198

Expression and roles of pannexins in ATP release in the pituitary gland.

PubMed

Li, Shuo; Bjelobaba, Ivana; Yan, Zonghe; Kucka, Marek; Tomic, Melanija; Stojilkovic, Stanko S

2011-06-01

Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

Factors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration, organelle integrity and ATP.

PubMed Central

Thomas, J; Debeer, L J; De Schepper, P J; Mannaerts, G P

1980-01-01

1. The first dehydrogenation step of peroxisomal beta-oxidation involves the reduction of O2 to H2O2. Production rates of H2O2 and acetyl units by purified rat liver peroxisomes oxidizing palmitoyl-CoA were equal, indicating that H2O2 production is a reliable index for the release of acetyl units during peroxisomal fatty-acid oxidation. 2. Measurements of H2O2 and acid-soluble oxidation products during [1-14C]palmitoyl-CoA oxidation by purified peroxisomes revealed that the number of acetyl units released per molecule of palmitoyl-CoA oxidized rapidly decreased with increasing unbound palmitoyl-CoA concentrations. Structural damage to the peroxisomes caused by detergents or other treatments also decreased the number of acetyl units released. Under conditions where oxidation proceeded linearly with time the theoretical maximum of 5 acetyl units released per molecule of palmitoyl-CoA oxidized [Lazarow (1978) J. Biol. Chem. 253, 1522--1528] was never reached. 3. Expressed in terms of acetyl units produced and measured at low unbound-palmitoyl-CoA concentrations, mitochondrial oxidation was 10--20-fold higher than peroxisomal oxidation. 4. ATP stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. The ATP effect required the presence of Mg2+ and was lost when peroxisomal membranes were disrupted by Triton X-100 or high concentrations of unbound palmitoyl-CoA. 5. Disruption of peroxisomes by detergents, freeze--thawing, osmotic or mechanical treatment did not stimulate palmitoyl-CoA oxidation in the presence of ATP, indicating that peroxisomal fatty-acid-CoA oxidation was not latent. In the absence of ATP, Triton X-100 stimulated peroxisomal palmitoyl-CoA oxidation approx. 2-fold. PMID:7470063

Regulation of blood flow distribution in skeletal muscle: role of erythrocyte-released ATP.

PubMed

Ellsworth, Mary L; Sprague, Randy S

2012-10-15

The maintenance of adequate tissue O(2) levels in skeletal muscle is vital for normal physiology and requires a well regulated and appropriately distributed convective O(2) supply. Inherent in this fundamental physiological process is the requirement for a mechanism which both senses tissue O(2) need and locally adjusts flow to appropriately meet that need. Over the past several years we and others have suggested that, in skeletal muscle, O(2) carrying erythrocytes participate in the regulation of total blood flow and its distribution by releasing ATP. Importantly, the release of this vasoactive molecule must be both rapid and well controlled if it is to serve an important physiological role. Here we provide insights into three distinct regulated signalling pathways within the erythrocyte that are activated by exposure to reduced O(2) tension or in response to binding of agonists to the prostacyclin or β-adrenergic receptors. Although much has been learned about the role of the erythrocyte in perfusion of skeletal muscle, much remains to be understood. However, what is clear is that the long established passive carrier of O(2) also contributes to the regulation of the distribution of microvascular perfusion in skeletal muscle by virtue of its capacity to release ATP.

Regulation of ACh release from guinea pig bladder urothelial cells: potential role in bladder filling sensations.

PubMed

McLatchie, L M; Young, J S; Fry, C H

2014-07-01

The aim of this study was to quantify and characterize the mechanism of non-neuronal ACh release from bladder urothelial cells and to determine if urothelial cells could be a site of action of anti-muscarinic drugs. A novel technique was developed whereby ACh could be measured from freshly isolated guinea pig urothelial cells in suspension following mechanical stimulation. Various agents were used to manipulate possible ACh release pathways in turn and to study the effects of muscarinic receptor activation and inhibition on urothelial ATP release. Minimal mechanical stimulus achieved full ACh release, indicating a small dynamic range and possible all-or-none signal. ACh release involved a mechanism dependent on the anion channel CFTR and intracellular calcium concentration, but was independent of extracellular calcium, vesicular trafficking, connexins or pannexins, organic cation transporters and was not affected by botulinum-A toxin. Stimulating ACh receptors increased ATP production and antagonizing them reduced ATP release, suggesting a link between ACh and ATP release. These results suggest that release of non-neuronal ACh from the urothelium is large enough and well located to act as a modulator of ATP release. It is hypothesized that this pathway may contribute to the actions of anti-muscarinic drugs in reducing the symptoms of lower urinary tract syndromes. Additionally the involvement of CFTR in ACh release suggests an exciting new direction for the treatment of these conditions. © 2014 The British Pharmacological Society.

Short-Term Exercise Training Does Not Stimulate Skeletal Muscle ATP Synthesis in Relatives of Humans With Type 2 Diabetes

PubMed Central

Kacerovsky-Bielesz, Gertrud; Chmelik, Marek; Ling, Charlotte; Pokan, Rochus; Szendroedi, Julia; Farukuoye, Michaela; Kacerovsky, Michaela; Schmid, Albrecht I.; Gruber, Stephan; Wolzt, Michael; Moser, Ewald; Pacini, Giovanni; Smekal, Gerhard; Groop, Leif; Roden, Michael

2009-01-01

OBJECTIVE We tested the hypothesis that short-term exercise training improves hereditary insulin resistance by stimulating ATP synthesis and investigated associations with gene polymorphisms. RESEARCH DESIGN AND METHODS We studied 24 nonobese first-degree relatives of type 2 diabetic patients and 12 control subjects at rest and 48 h after three bouts of exercise. In addition to measurements of oxygen uptake and insulin sensitivity (oral glucose tolerance test), ectopic lipids and mitochondrial ATP synthesis were assessed using1H and31P magnetic resonance spectroscopy, respectively. They were genotyped for polymorphisms in genes regulating mitochondrial function, PPARGC1A (rs8192678) and NDUFB6 (rs540467). RESULTS Relatives had slightly lower (P = 0.012) insulin sensitivity than control subjects. In control subjects, ATP synthase flux rose by 18% (P = 0.0001), being 23% higher (P = 0.002) than that in relatives after exercise training. Relatives responding to exercise training with increased ATP synthesis (+19%, P = 0.009) showed improved insulin sensitivity (P = 0.009) compared with those whose insulin sensitivity did not improve. A polymorphism in the NDUFB6 gene from respiratory chain complex I related to ATP synthesis (P = 0.02) and insulin sensitivity response to exercise training (P = 0.05). ATP synthase flux correlated with O2uptake and insulin sensitivity. CONCLUSIONS The ability of short-term exercise to stimulate ATP production distinguished individuals with improved insulin sensitivity from those whose insulin sensitivity did not improve. In addition, the NDUFB6 gene polymorphism appeared to modulate this adaptation. This finding suggests that genes involved in mitochondrial function contribute to the response of ATP synthesis to exercise training. PMID:19265027

FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

PubMed

Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

2013-02-01

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. Copyright © 2012 Wiley Periodicals, Inc.

The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling.

PubMed

Losano, João D A; Padín, Juan Fernando; Méndez-López, Iago; Angrimani, Daniel S R; García, Antonio G; Barnabe, Valquiria H; Nichi, Marcilio

2017-01-01

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specificallymore » with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. In conclusion, taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.« less

Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

DOE PAGES

Epling, Leslie B.; Grace, Christy R.; Lowe, Brandon R.; ...

2015-02-25

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specificallymore » with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. In conclusion, taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.« less

Effect of botulinum toxin A on urothelial-release of ATP and expression of SNARE targets within the urothelium.

PubMed

Hanna-Mitchell, Ann T; Wolf-Johnston, Amanda S; Barrick, Stacey R; Kanai, Anthony J; Chancellor, Michael B; de Groat, William C; Birder, Lori A

2015-01-01

Botulinum neurotoxin serotype A (BoNT/A) has emerged as an effective treatment of urinary bladder overactivity. Intravesical lipotoxin (BoNT/A delivery using liposomes), which may target the urothelium, is effective in blocking acetic acid induced hyperactivity in animals. The objective of this study was to assess the possible site of toxin action within the urothelium. We examined expression of the toxin receptor (SV2) and its cleavage targets (SNAP-25 and SNAP-23) within urothelium as well as effects of the toxin on mechanically evoked release of ATP from cultured rat urothelial cells. ATP release was measured using the luciferin-luciferase assay; we examined expression of SNAP-23 and -25 in urothelial cells and mucosa of rat and human bladders. BoNT/A (1.5 U; 1-3 hr) blocked hypotonic evoked release of urothelial ATP, without affecting morphology. The expression of protein targets for BoNT/A binding (SV2) was detected in human and rat bladder mucosa and catalytic action (SNAP-23, -25) in urothelial cells and mucosa (differed in intensity) from rat and human bladder. Incubation of cultured (rat) urothelial cells with BoNT/A decreased expression levels of both SNAP-23 (44%) and SNAP-25 (80%). Our findings reveal that the bladder urothelium expresses the intracellular targets and the binding protein for cellular uptake of BoNT/A; and that the toxin is able to suppress the levels of these targets as well as hypotonic-evoked ATP release. These data raise the possibility that intravesical treatment with BoNT/A suppresses bladder reflex and sensory mechanisms by affecting a number of urothelial functions including release of transmitters. © 2013 Wiley Periodicals, Inc.

Mechanosensitive ATP Release Maintains Proper Mucus Hydration of Airways

PubMed Central

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

2013-01-01

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

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

PubMed Central

Suzuki, Kano; Mizutani, Kenji; Maruyama, Shintaro; Shimono, Kazumi; Imai, Fabiana L.; Muneyuki, Eiro; Kakinuma, Yoshimi; Ishizuka-Katsura, Yoshiko; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamato, Ichiro; Murata, Takeshi

2016-01-01

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model. PMID:27807367

Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays.

PubMed

Ohshima, Yasuhiro; Kitami, Akihiro; Kawano, Ayumi; Tsukimoto, Mitsutoshi; Kojima, Shuji

2011-09-15

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

Glucose recruits K(ATP) channels via non-insulin-containing dense-core granules.

PubMed

Yang, Shao-Nian; Wenna, Nancy Dekki; Yu, Jia; Yang, Guang; Qiu, Hua; Yu, Lina; Juntti-Berggren, Lisa; Köhler, Martin; Berggren, Per-Olof

2007-09-01

beta cells rely on adenosine triphosphate-sensitive potassium (K(ATP)) channels to initiate and end glucose-stimulated insulin secretion through changes in membrane potential. These channels may also act as a constituent of the exocytotic machinery to mediate insulin release independent of their electrical function. However, the molecular mechanisms whereby the beta cell plasma membrane maintains an appropriate number of K(ATP) channels are not known. We now show that glucose increases K(ATP) current amplitude by increasing the number of K(ATP) channels in the beta cell plasma membrane. The effect was blocked by inhibition of protein kinase A (PKA) as well as by depletion of extracellular or intracellular Ca(2+). Furthermore, glucose promoted recruitment of the potassium inward rectifier 6.2 to the plasma membrane, and intracellular K(ATP) channels localized in chromogranin-positive/insulin-negative dense-core granules. Our data suggest that glucose can recruit K(ATP) channels to the beta cell plasma membrane via non-insulin-containing dense-core granules in a Ca(2+)- and PKA-dependent manner.

Gastrin-releasing peptide stimulates glycoconjugate release from feline trachea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundgren, J.D.; Baraniuk, J.N.; Ostrowski, N.L.

1990-02-01

The effect of gastrin-releasing peptide (GRP) on respiratory glycoconjugate (RGC) secretion was investigated in a feline tracheal organ culture model. RGC secretion was stimulated by GRP in a dose-dependent fashion at concentrations from 10(-8) to 10(-5) M (range 15-38% increase above control) with a peak effect within 0.5-1 h of incubation. GRP-(14-27), the receptor binding portion of GRP, and the related molecule, bombesin, also stimulated RGC secretion by approximately 20% above control. Acetyl-GRP-(20-27) stimulated RGC release by 10%, whereas GRP-(1-16) was inactive. Autoradiographic studies with 125I-GRP revealed that specific binding was restricted to the submucosal glands and the surface epithelium.more » A specific radioimmunoassay showed the content of GRP in feline trachea after extraction with ethanol-acetic acid to be 156 +/- 91 fmol/g wet wt. Indirect immunohistochemistry indicated that ganglion cells located just outside the cartilage contained GRP-immunoreactive materials. GRP is a novel mucus secretagogue that may participate in regulating airway mucosal gland secretion.« less

Mechanical loading stimulates ecto-ATPase activity in human tendon cells.

PubMed

Tsuzaki, M; Bynum, D; Almekinders, L; Faber, J; Banes, A J

2005-09-01

Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5'-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor-ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5'-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5-1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD

Hydrolysis of Extracellular ATP by Ectonucleoside Triphosphate Diphosphohydrolase (ENTPD) Establishes the Set Point for Fibrotic Activity of Cardiac Fibroblasts*

PubMed Central

Lu, David; Insel, Paul A.

2013-01-01

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs. PMID:23677997

In vitro release of adenosine triphosphate from the urothelium of human bladders with detrusor overactivity, both neurogenic and idiopathic.

PubMed

Kumar, Vivek; Chapple, Christopher R; Rosario, Derek; Tophill, Paul R; Chess-Williams, Russell

2010-06-01

There is increased evidence to suggest a role for nonadrenergic-noncholinergic neurotransmission in the pathogenesis of bladder dysfunction. In this set of experiments, we have assessed the contribution of the urothelium to purinergic activity by quantifying the amount of adenosine triphosphate (ATP) released from the urothelium of patients with idiopathic detrusor overactivity (IDO) and with neurogenic detrusor overactivity (NDO) and comparing these releases to those of controls. Bladder tissue with urodynamically and clinically proven NDO (n=8) and IDO (n=8) were included in this study. The carefully dissected urothelium was stimulated by mechanically stretching as well as electrically stimulating and the ATP; thus, release was quantified. We used a Lucy Anthos 1 luminometre (Anthos Labtec Instruments GmBH, Wals, Austria) to perform the assay. The results were analysed using Stingray software (Dazdaq Ltd, Brighton, UK). Both mechanical stretch and electric field stimulation (EFS) led to increased ATP release in both sets of tissues with overactivity compared to the controls; this rise was even more significant for the IDO urothelium (2416.7±479.8 pmol/g [p<0.005]) than for the NDO urothelium (133.1±22.4 pmol/g [p<0.01]); values for the controls were 77.6±16.2 pmol/g. ATP release following mechanical stretch was more sensitive to tetrodotoxin in bladders with NDO compared to those with IDO as well as to the controls, with ATP levels falling from 233.5±20.7 pmol/g to 107.2±11.6 pmol/g, expressed as percentage of basal levels (p<0.002). The experiments were performed in vitro, and the female patients were a mix of peri- and postmenopausal states. These experiments suggested a significant rise in ATP release from the urothelium of bladders with NDO as well as those with IDO in comparison to controls. Most of the ATP released from bladders with NDO is primarily from neuronal sources. Copyright © 2009 European Association of Urology. Published by Elsevier B

Direct stimulation of pituitary prolactin release by glutamate.

PubMed

Login, I S

1990-01-01

The ability of glutamate and other excitatory amino acids to stimulate prolactin secretion when administered to adult animals is hypothesized to depend on a central site of action in the brain, but there are no data to support this position. An alternative hypothesis was tested that glutamate would stimulate prolactin release when applied directly to primary cultures of dispersed adult female rat anterior pituitary cells studied in a perifusion protocol. Glutamate increased the rate of prolactin release within two minutes in a self-limited manner. Glutamate-stimulated prolactin release was augmented about 4-fold by elimination of magnesium from the perfusate and was associated with stimulation of pituitary calcium flux. Ketamine and MK-801 both reduced the basal rate of prolactin release and abolished the effects of glutamate. Pituitary cells of 10-day-old rats responded similarly to glutamate. Exposure to glutamate did not influence subsequent responses to physiological hypothalamic secretagogues, thus the likelihood of toxicity was minimized. These results suggest that the N-methyl-D-aspartate (NMDA) subclass of the glutamate receptor complex is involved. Prolactin secretion may be regulated physiologically through a functional glutamate receptor on pituitary cells.

The Lumenal Loop Met672–Pro707 of Copper-transporting ATPase ATP7A Binds Metals and Facilitates Copper Release from the Intramembrane Sites*

PubMed Central

Barry, Amanda N.; Otoikhian, Adenike; Bhatt, Sujata; Shinde, Ujwal; Tsivkovskii, Ruslan; Blackburn, Ninian J.; Lutsenko, Svetlana

2011-01-01

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met672–Pro707 (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ∼2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)2 site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed. PMID:21646353

Computational Modeling of Neurotransmitter Release Evoked by Electrical Stimulation: Nonlinear Approaches to Predicting Stimulation-Evoked Dopamine Release.

PubMed

Trevathan, James K; Yousefi, Ali; Park, Hyung Ook; Bartoletta, John J; Ludwig, Kip A; Lee, Kendall H; Lujan, J Luis

2017-02-15

Neurochemical changes evoked by electrical stimulation of the nervous system have been linked to both therapeutic and undesired effects of neuromodulation therapies used to treat obsessive-compulsive disorder, depression, epilepsy, Parkinson's disease, stroke, hypertension, tinnitus, and many other indications. In fact, interest in better understanding the role of neurochemical signaling in neuromodulation therapies has been a focus of recent government- and industry-sponsored programs whose ultimate goal is to usher in an era of personalized medicine by creating neuromodulation therapies that respond to real-time changes in patient status. A key element to achieving these precision therapeutic interventions is the development of mathematical modeling approaches capable of describing the nonlinear transfer function between neuromodulation parameters and evoked neurochemical changes. Here, we propose two computational modeling frameworks, based on artificial neural networks (ANNs) and Volterra kernels, that can characterize the input/output transfer functions of stimulation-evoked neurochemical release. We evaluate the ability of these modeling frameworks to characterize subject-specific neurochemical kinetics by accurately describing stimulation-evoked dopamine release across rodent (R 2 = 0.83 Volterra kernel, R 2 = 0.86 ANN), swine (R 2 = 0.90 Volterra kernel, R 2 = 0.93 ANN), and non-human primate (R 2 = 0.98 Volterra kernel, R 2 = 0.96 ANN) models of brain stimulation. Ultimately, these models will not only improve understanding of neurochemical signaling in healthy and diseased brains but also facilitate the development of neuromodulation strategies capable of controlling neurochemical release via closed-loop strategies.

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. © Georg Thieme Verlag KG Stuttgart · New York.

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons.

PubMed

Login, I S; Pal, S N; Adams, D T; Gold, P E

1998-01-01

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [3H]ACh release was recorded during perifusion of acutely dissociated, [3H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally approximately 300% with EC50 = approximately 1 microM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K+ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

PubMed

Modali, Sita D; Zgurskaya, Helen I

2011-08-01

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins. © 2011 Blackwell Publishing Ltd.

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter

PubMed Central

Modali, Sita D.; Zgurskaya, Helen I.

2011-01-01

Escherichia coli MacAB-TolC is a tri-partite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins. PMID:21696464

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

PubMed

MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

2007-06-01

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Insulin-releasing action of the novel antidiabetic agent BTS 67 582.

PubMed

McClenaghan, N H; Flatt, P R; Bailey, C J

1998-02-01

1. BTS 67582 (1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate) is a novel antidiabetic agent with a short-acting insulin-releasing effect. This study examined its mode of action in the clonal B-cell line BRIN-BD11. 2. BTS 67582 increased insulin release from BRIN-BD11 cells in a concentration-dependent manner (10[-8] to 10[-4] M) at both non-stimulating (1.1 mM) and stimulating (16.7 mM) concentrations of glucose. 3. BTS 67582 (10[-4] M) potentiated the insulin-releasing effect of a depolarizing concentration of K+ (30 mM), whereas the K+ channel openers pinacidil (400 microM) and diazoxide (300 microM) inhibited BTS 67582-induced release. 4. Suppression of Ca+ channel activity with verapamil (20 microM) reduced the insulin-releasing effect of BTS 67582 (10[-4] M). 5. BTS 67582 (10[-4] M) potentiated insulin release induced by amino acids (10 mM), and enhanced the combined stimulant effects of glucose plus either the fatty acid palmitate (10 mM), or agents which raise intracellular cyclic AMP concentrations (25 microM forskolin and 1 mM isobutylmethylxanthine), or the cholinoceptor agonist carbachol (100 microM). 6. Inhibition of glucose-stimulated insulin release by adrenaline or noradrenaline (10 microM) was partially reversed by BTS 67582 (10[-4] M). 7. These data suggest that the insulin-releasing effect of BTS 67582 involves regulation of ATP-sensitive K+ channel activity and Ca2+ influx, and that the drug augments the stimulant effects of nutrient insulin secretagogues and agents which enhance adenylate cyclase and phospholipase C. BTS 67582 may also exert insulin-releasing effects independently of ATP-sensitive K+ channel activity.

Red blood cell dynamics: from cell deformation to ATP release.

PubMed

Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

2011-10-01

The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release. This journal is © The Royal Society of Chemistry 2011

Ultrasound Stimulation of Insulin Release from Pancreatic Beta Cells

NASA Astrophysics Data System (ADS)

Suarez Castellanos, Ivan M.

Type 2 diabetes (T2D) mellitus is a complex metabolic disease that has reached epidemic proportions in the United States and around the world. Controlling T2D is often difficult as pharmacological management routinely requires complex therapy with multiple medications, and loses its effectiveness over time. The objective of this dissertation was to explore a novel, non-pharmacological approach that utilizes the application of ultrasound energy to stimulate insulin release. Our experiments have focused on determination of effectiveness and safety of ultrasound application in stimulation of insulin release from the pancreatic beta cells. Our results showed that ultrasound treatment, applied at frequencies of 800 kHz and 1 MHz and intensities of 0.5 W/cm2 and 1 W/cm2, did not produce any significant effects on cell viability compared to sham group as assessed with trypan blue dye exclusion test and MTT cytotoxicity assay. ELISA quantification of insulin release from beta cells resulting from ultrasound treatment showed clinically-significant amounts of released insulin as compared to sham-treated beta cells. Carbon fiber amperometry detection of secretory events from dopamine-loaded beta cells treated with ultrasound showed that release of secretory content could be temporally controlled by careful selection of ultrasound parameters. Both ELISA and amperometry experiments demonstrated that ultrasound-stimulated insulin release is a calcium-dependent process, potentially mediated by the mechanical effects of ultrasound. This study demonstrated that therapeutic ultrasound is a technique capable of stimulating the release of insulin from pancreatic beta cells in a safe, effective and controlled manner.

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction.

PubMed

Chandramouli, Kala; Johnson, Michael K

2006-09-19

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.

Diadenosine polyphosphate-stimulated gluconeogenesis in isolated rat proximal tubules.

PubMed Central

Edgecombe, M; Craddock, H S; Smith, D C; McLennan, A G; Fisher, M J

1997-01-01

Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP. PMID:9163337

A novel inositol phosphate selectively inhibits vasoconstriction evoked by the sympathetic co-transmitters neuropeptide Y (NPY) and adenosine triphosphate (ATP).

PubMed

Wahlestedt, C; Reis, D J; Yoo, H; Adamsson, M; Andersson, D; Edvinsson, L

1992-08-31

Postganglionic sympathetic nerves release norepinephrine (NE) as their primary neurotransmitter at vascular and other targets. However, much evidence supports involvement of additional messengers, co-transmitters, which are co-released with NE upon sympathetic nerve stimulation and thereby contribute to their actions, e.g., vasoconstriction. Two such putative co-transmitters, neuropeptide Y (NPY) and adenosine triphosphate (ATP) have been of particular interest since they fulfill several neurotransmitter criteria. Importantly, hitherto it has been difficult to antagonize vasoconstriction evoked by either NPY or ATP with agents that are devoid of intrinsic activity. The present study describes the ability of a novel inositol phosphate, D-myo-inositol 1,2,6-trisphosphate (Ins[1,2,6]P3; PP-56) to in vitro potently block vasoconstrictor responses elicited by NPY and ATP, but not by NE, as studied in guinea-pig isolated basilar artery. The action of Ins[1,2,6]P3 does not seem to occur through antagonism at NPY- or ATP-receptor recognition sites, labeled by 125I-peptide YY and 35S-gamma-ATP, respectively, in membranes of rat cultured vena cava vascular smooth muscle cells. However, it does involve inhibition of the influx of Ca2+ induced by either co-transmitter in these same vena cava cells. It is proposed that Ins[1,2,6]P3 may be a useful functional antagonist of non-adrenergic component(s) of the vasoconstrictor response to sympathetic nerve stimulation.

Canine bombesin-like gastrin releasing peptides stimulate gastrin release and acid secretion in the dog.

PubMed Central

Bunnett, N W; Clark, B; Debas, H T; Del Milton, R C; Kovacs, T O; Orloff, M S; Pappas, T N; Reeve, J R; Rivier, J E; Walsh, J H

1985-01-01

The synthetic mammalian bombesin-like peptides, canine gastrin releasing peptide 27, 23 and 10, and porcine gastrin releasing peptide 27 were compared with amphibian bombesin 14 and 10 during intravenous infusions into six conscious dogs with chronic gastric cannulae. Gastrin and gastrin releasing peptide were measured in peripherally sampled venous blood by radioimmunoassay and gastric acid secretions were collected. All forms of gastrin releasing peptide stimulated gastrin release and gastric acid secretion in a dose-dependent manner. The larger canine and porcine peptides were more potent than the decapeptide. Bombesin 14 was more potent than bombesin 10. A rise in the venous concentration of immunoreactive gastrin releasing peptide of only 20 fmol ml-1 stimulated gastrin release to about 50% of maximal. Gastrin releasing peptide 10 was cleared from the circulation three times faster than the larger forms and this may account for the apparent differences in potency. PMID:3839849

ATP excites mouse vomeronasal sensory neurons through activation of P2X receptors.

PubMed

Vick, J S; Delay, R J

2012-09-18

Purinergic signaling through activation of P2X and P2Y receptors is critically important in the chemical senses. In the mouse main olfactory epithelium (MOE), adenosine 5'-triphosphate (ATP) elicits an increase in intracellular calcium ([Ca(2+)](I)) and reduces the responsiveness of olfactory sensory neurons to odorants through activation of P2X and P2Y receptors. We investigated the role of purinergic signaling in vomeronasal sensory neuron (VSN)s from the mouse vomeronasal organ (VNO), an olfactory organ distinct from the MOE that responds to many conspecific chemical cues. Using a combination of calcium imaging and patch-clamp electrophysiology with isolated VSNs, we demonstrated that ATP elicits an increase in [Ca(2+)](I) and an inward current with similar EC(50)s. Neither adenosine nor the P2Y receptor ligands adenosine 5'-diphosphate, uridine 5'-triphosphate, and uridine-5'-disphosphate could mimic either effect of ATP. Moreover, the increase in [Ca(2+)](I) required the presence of extracellular calcium and the inward current elicited by ATP was partially blocked by the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate. Consistent with the activation of P2X receptors, we detected gene expression of the P2X1 and 3 receptors in the VNO by Reverse transcription polymerase chain reaction (RT-PCR). When co-delivered with dilute urine, a natural stimulus, ATP significantly increased the inward current above that elicited by dilute urine or ATP alone. Mechanical stimulation of the VNO induced the release of ATP, detected by luciferin-luciferase luminometry, and this release of ATP was completely abolished in the presence of the connexin/pannexin hemichannel blocker, carbenoxolone. We conclude that the release of ATP could occur during the activity of the vasomotor pump that facilitates the movement of chemicals into the VNO for detection by VSNs. This mechanism could lead to a

Quantifying Ca2+ release and inactivation of Ca2+ release in fast- and slow-twitch muscles.

PubMed

Barclay, C J

2012-12-01

The aims of this study were to quantify the Ca(2+) release underlying twitch contractions of mammalian fast- and slow-twitch muscle and to comprehensively describe the transient inactivation of Ca(2+) release following a stimulus. Experiments were performed using bundles of fibres from mouse extensor digitorum longus (EDL) and soleus muscles. Ca(2+) release was quantified from the amount of ATP used to remove Ca(2+) from the myoplasm following stimulation. ATP turnover by crossbridges was blocked pharmacologically (N-benzyl-p-toluenesulphonamide for EDL, blebbistatin for soleus) and muscle heat production was used as an index of Ca(2+) pump ATP turnover. At 20°C, Ca(2+) release in response to a single stimulus was 34 and 84 μmol (kg muscle)(-1) for soleus and EDL, respectively, and increased with temperature (30°C: soleus, 61 μmol kg(-1); EDL, 168 μmol kg(-1)). Delivery of another stimulus within 100 ms of the first produced a smaller Ca(2+) release. The maximum magnitude of the decrease in Ca(2+) release was greater in EDL than soleus. Ca(2+) release recovered with an exponential time course which was faster in EDL (mean time constant at 20°C, 32.1 ms) than soleus (65.6 ms) and faster at 30°C than at 20°C. The amounts of Ca(2+) released and crossbridge cycles performed are consistent with a scheme in which Ca(2+) binding to troponin-C allowed an average of ∼1.7 crossbridge cycles in the two muscles.

Quantifying Ca2+ release and inactivation of Ca2+ release in fast- and slow-twitch muscles

PubMed Central

Barclay, C J

2012-01-01

The aims of this study were to quantify the Ca2+ release underlying twitch contractions of mammalian fast- and slow-twitch muscle and to comprehensively describe the transient inactivation of Ca2+ release following a stimulus. Experiments were performed using bundles of fibres from mouse extensor digitorum longus (EDL) and soleus muscles. Ca2+ release was quantified from the amount of ATP used to remove Ca2+ from the myoplasm following stimulation. ATP turnover by crossbridges was blocked pharmacologically (N-benzyl-p-toluenesulphonamide for EDL, blebbistatin for soleus) and muscle heat production was used as an index of Ca2+ pump ATP turnover. At 20°C, Ca2+ release in response to a single stimulus was 34 and 84 μmol (kg muscle)−1 for soleus and EDL, respectively, and increased with temperature (30°C: soleus, 61 μmol kg−1; EDL, 168 μmol kg−1). Delivery of another stimulus within 100 ms of the first produced a smaller Ca2+ release. The maximum magnitude of the decrease in Ca2+ release was greater in EDL than soleus. Ca2+ release recovered with an exponential time course which was faster in EDL (mean time constant at 20°C, 32.1 ms) than soleus (65.6 ms) and faster at 30°C than at 20°C. The amounts of Ca2+ released and crossbridge cycles performed are consistent with a scheme in which Ca2+ binding to troponin-C allowed an average of ∼1.7 crossbridge cycles in the two muscles. PMID:23027818

ATP Releasing Connexin 30 Hemichannels Mediate Flow-Induced Calcium Signaling in the Collecting Duct

PubMed Central

Svenningsen, Per; Burford, James L.; Peti-Peterdi, János

2013-01-01

ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y2 receptor, ENaC, and AQP2. Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs ([Ca2+]i increased only 1.2-fold, p < 0.0001 vs. WT, n = 6 each). To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption. PMID:24137132

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

Regulated release of ERdj3 from unfolded proteins by BiP

PubMed Central

Jin, Yi; Awad, Walid; Petrova, Kseniya; Hendershot, Linda M

2008-01-01

DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that were defective in any step of the ATPase cycle were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP's ATPase activity are required to release ERdj3 from substrate and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70–DnaJ pairs. PMID:18923428

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apo-ferredoxin in an ATP-dependent reaction†

PubMed Central

Chandramouli, Kala; Johnson, Michael K.

2008-01-01

The role of the Azotobacter vinelandii HscA/HscB co-chaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S] IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulated HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB co-chaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU (Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931), and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed. PMID:16964969

UTP - Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells.

PubMed

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca 2+ -dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca 2+ imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca 2+ oscillations in BON. UTP evoked a biphasic concentration-dependent Ca 2+ response. Cells responded in the order of UTP, ATP > UTPγS > UDP > MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y 4 , 50% cells), UDP (P2Y 6 , 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca 2+ responses were blocked with inhibitors of PLC, IP3R, SERCA Ca 2+ pump, La 3+ sensitive Ca 2+ channels or chelation of intracellular free Ca 2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca 2+ pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca 2+ currents (I Ca ), V m -depolarization and inhibited I K (not I A ) currents. An I Kv 7.2/7.3 K + channel blocker XE-991 mimicked UTP-induced V m -depolarization and blocked UTP-responses. XE-991 blocked I K and UTP caused further reduction. La 3+ or PLC inhibitors blocked UTP depolarization

P-glycoprotein interactions of novel psychoactive substances - stimulation of ATP consumption and transport across Caco-2 monolayers.

PubMed

Meyer, Markus R; Wagmann, Lea; Schneider-Daum, Nicole; Loretz, Brigitta; de Souza Carvalho, Cristiane; Lehr, Claus-Michael; Maurer, Hans H

2015-04-01

In contrast to drugs for therapeutic use, there are only few data available concerning interactions between P-glycoprotein (P-gp) and drugs of abuse (DOA). In this work, interactions between structurally diverse DOA and P-gp were investigated using different strategies. First, the effect on the P-gp ATPase activity was studied by monitoring of ATP consumption after addition to recombinant, human P-gp. Second, DOA showing an increased ATP consumption were further characterized regarding their transport across filter grown Caco-2- monolayers. Analyses were performed by luminescence and liquid chromatography-mass spectrometry, respectively. Among the nine DOA initially screened, benzedrone, diclofensine, glaucine, JWH-200, MDBC, WIN-55,212-2 showed an increase of ATP consumption in the ATPase stimulation assay. In Caco-2 transport studies, Glaucine, JWH-200, mitragynine, WIN-55,212-2 could moreover be identified as non-transported substrates, but inhibitors of P-gp activity. Thus, drug-drug or drug-food interactions should be very likely for these compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Significance of air humidity and air velocity for fungal spore release into the air

NASA Astrophysics Data System (ADS)

Pasanen, A.-L.; Pasanen, P.; Jantunen, M. J.; Kalliokoski, P.

Our previous field studies have shown that the presence of molds in buildings does not necessarily mean elevated airborne spore counts. Therefore, we investigated the release of fungal spores from cultures of Aspergillus fumigatus, Penicillium sp. and Cladosporium sp. at different air velocities and air humidities. Spores of A. fumigatus and Penicillium sp. were released from conidiophores already at air velocity of 0.5 ms -1, whereas Cladosporium spores required at least a velocity of 1.0 ms -1. Airborne spore counts of A. fumigatus and Penicillium sp. were usually higher in dry than moist air, being minimal at relative humidities (r.h.) above 70%, while the effect of r.h. on the release of Cladosporium sp. was ambivalent. The geometric mean diameter of released spores increased when the r.h. exceeded a certain level which depends on fungal genus. Thus, spores of all three fungi were hygroscopic but the hygroscopicity of various spores appeared at different r.h.-ranges. This study indicates that spore release is controlled by external factors and depends on fungal genus which can be one reason for considerable variation of airborne spore counts in buildings with mold problems.

The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically

PubMed Central

Richardson, I.W.; Szerb, J.C.

1974-01-01

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [3H]-choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation. 2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [3H]-ACh which was greatly enhanced by the addition of atropine. 3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [3H]-ACh which declined asymptotically. This evoked release appeared to follow first-order kinetics with a rate constant which remained stable over the course of prolonged stimulation. 4 The rate constant for the evoked release of [3H]-ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [3H]-ACh was released was nearly identical under these two conditions. 5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [3H]-choline in the samples. 6 Reduction of [3H]-ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [3H]-ACh without affecting the release of [3H]-choline. 7 Results suggest that electrical stimulation of cortical slices which had been incubated with [3H]-choline causes the release of only [3H]-ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [3H]-ACh. PMID:4455326

UTP – Gated Signaling Pathways of 5-HT Release from BON Cells as a Model of Human Enterochromaffin Cells

PubMed Central

Liñán-Rico, Andromeda; Ochoa-Cortes, Fernando; Zuleta-Alarcon, Alix; Alhaj, Mazin; Tili, Esmerina; Enneking, Josh; Harzman, Alan; Grants, Iveta; Bergese, Sergio; Christofi, Fievos L.

2017-01-01

Background: Enterochromaffin cells (EC) synthesize and release 5-HT and ATP to trigger or modulate gut neural reflexes and transmit information about visceral/pain sensation. Alterations in 5-HT signaling mechanisms may contribute to the pathogenesis of IBD or IBS, but the pharmacologic or molecular mechanisms modulating Ca2+-dependent 5-HT release are not understood. Previous studies indicated that purinergic signaling via ATP and ADP is an important mechanism in modulation of 5-HT release. However, EC cells also respond to UTP and UDP suggesting uridine triphosphate receptor and signaling pathways are involved as well. We tested the hypothesis that UTP is a regulator of 5-HT release in human EC cells. Methods: UTP signaling mechanisms were studied in BON cells, a human EC model, using Fluo-4/Ca2+imaging, patch-clamp, pharmacological analysis, immunohistochemistry, western blots and qPCR. 5-HT release was monitored in BON or EC isolated from human gut surgical specimens (hEC). Results: UTP, UTPγS, UDP or ATP induced Ca2+oscillations in BON. UTP evoked a biphasic concentration-dependent Ca2+response. Cells responded in the order of UTP, ATP > UTPγS > UDP >> MRS2768, BzATP, α,β-MeATP > MRS2365, MRS2690, and NF546. Different proportions of cells activated by UTP and ATP also responded to UTPγS (P2Y4, 50% cells), UDP (P2Y6, 30%), UTPγS and UDP (14%) or MRS2768 (<3%). UTP Ca2+responses were blocked with inhibitors of PLC, IP3R, SERCA Ca2+pump, La3+sensitive Ca2+channels or chelation of intracellular free Ca2+ by BAPTA/AM. Inhibitors of L-type, TRPC, ryanodine-Ca2+pools, PI3-Kinase, PKC or SRC-Kinase had no effect. UTP stimulated voltage-sensitive Ca2+currents (ICa), Vm-depolarization and inhibited IK (not IA) currents. An IKv7.2/7.3 K+ channel blocker XE-991 mimicked UTP-induced Vm-depolarization and blocked UTP-responses. XE-991 blocked IK and UTP caused further reduction. La3+ or PLC inhibitors blocked UTP depolarization; PKC inhibitors, thapsigargin or zero Ca

Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release

PubMed Central

Melchior, James R.; Ferris, Mark J.; Stuber, Garret D.; Riddle, David R.; Jones, Sara R.

2015-01-01

The nucleus accumbens is highly heterogeneous, integrating regionally distinct afferent projections and accumbal interneurons, resulting in diverse local microenvironments. Dopamine (DA) neuron terminals similarly express a heterogeneous collection of terminal receptors that modulate DA signaling. Cyclic voltammetry is often used to probe DA terminal dynamics in brain slice preparations; however, this method traditionally requires electrical stimulation to induce DA release. Electrical stimulation excites all of the neuronal processes in the stimulation field, potentially introducing simultaneous, multi-synaptic modulation of DA terminal release. We used optogenetics to selectively stimulate DA terminals and used voltammetry to compare DA responses from electrical and optical stimulation of the same area of tissue around a recording electrode. We found that with multiple pulse stimulation trains, optically stimulated DA release increasingly exceeded that of electrical stimulation. Furthermore, electrical stimulation produced inhibition of DA release across longer duration stimulations. The GABAB antagonist, CGP 55845, increased electrically stimulated DA release significantly more than light stimulated release. The nicotinic acetylcholine receptor antagonist, dihydro-β-erythroidine hydrobromide, inhibited single pulse electrically stimulated DA release while having no effect on optically stimulated DA release. Our results demonstrate that electrical stimulation introduces local multi-synaptic modulation of DA release that is absent with optogenetically targeted stimulation. PMID:26011081

Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S-21663: evidence for the existence of a novel imidazoline site in β cells

PubMed Central

Le Brigand, Laurence; Virsolvy, Anne; Peyrollier, Karine; Manechez, Dominique; Godfroid, Jean-Jacques; Guardiola-Lemaître, Béatrice; Bataille, Dominique

1997-01-01

The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic β cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas. S-21663, at concentrations ranging from 10−5 M to 10−3 M was able to release insulin from MIN6 cells; its activity peaked at 10−4 M, a drop in the stimulant factor being noted between 10−4 and 10−3 M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1. In contrast to tGLP-1, S-21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil- and nifedipine-sensitive voltage-dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S-21663-induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP-dependent K+ (K-ATP) channels as determined by measurement of 86Rb efflux and use of a K-ATP channel opener. Comparison of the effects of S-21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose-dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S-21663 acts through a novel site which displays a remarkably stable expression along the cell culture. It is concluded that S-21663 is a very efficient, glucose-independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and ‘efaroxan' binding sites. In vitro and in vivo features of S-21663 indicate that this compound, or new drugs drived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin-dependent) diabetes. PMID:9375978

Acetylcholine is released from taste cells, enhancing taste signalling

PubMed Central

Dando, Robin; Roper, Stephen D

2012-01-01

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

ATP synthesis in Halobacterium saccharovorum: evidence that synthesis may be catalysed by an F0F1-ATP synthase

NASA Technical Reports Server (NTRS)

Hochstein, L. I.

1992-01-01

Halobacterium saccharovorum synthesized ATP in response to a pH shift from 8 to 6.2. Synthesis was inhibited by carbonyl cyanide m-chloro-phenylhydrazone, dicyclohexylcarbodiimide, and azide. Nitrate, an inhibitor of the membrane-bound ATPase previously isolated from this organism, did not inhibit ATP synthesis. N-Ethymaleimide, which also inhibited this ATPase, stimulated the production of ATP. These observations suggested that H. saccharovorum synthesized and hydrolysed ATP using different enzymes and that the vacuolar-like ATPase activity previously described in H. saccharovorum was an ATPase whose function is yet to be identified.

21 CFR 874.1800 - Air or water caloric stimulator.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Air or water caloric stimulator. 874.1800 Section 874.1800 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES EAR, NOSE, AND THROAT DEVICES Diagnostic Devices § 874.1800 Air or water caloric stimulator. (a) Identification. An air or wate...

Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

PubMed Central

Ah-Seng, Yoan; Rech, Jérôme; Lane, David; Bouet, Jean-Yves

2013-01-01

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability. PMID:24367270

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain.

PubMed

Kato, Yuri; Hiasa, Miki; Ichikawa, Reiko; Hasuzawa, Nao; Kadowaki, Atsushi; Iwatsuki, Ken; Shima, Kazuhiro; Endo, Yasuo; Kitahara, Yoshiro; Inoue, Tsuyoshi; Nomura, Masatoshi; Omote, Hiroshi; Moriyama, Yoshinori; Miyaji, Takaaki

2017-07-18

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl - A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

Nutritional State-Dependent Ghrelin Activation of Vasopressin Neurons via Retrograde Trans-Neuronal–Glial Stimulation of Excitatory GABA Circuits

PubMed Central

Haam, Juhee; Halmos, Katalin C.; Di, Shi

2014-01-01

Behavioral and physiological coupling between energy balance and fluid homeostasis is critical for survival. The orexigenic hormone ghrelin has been shown to stimulate the secretion of the osmoregulatory hormone vasopressin (VP), linking nutritional status to the control of blood osmolality, although the mechanism of this systemic crosstalk is unknown. Here, we show using electrophysiological recordings and calcium imaging in rat brain slices that ghrelin stimulates VP neurons in the hypothalamic paraventricular nucleus (PVN) in a nutritional state-dependent manner by activating an excitatory GABAergic synaptic input via a retrograde neuronal–glial circuit. In slices from fasted rats, ghrelin activation of a postsynaptic ghrelin receptor, the growth hormone secretagogue receptor type 1a (GHS-R1a), in VP neurons caused the dendritic release of VP, which stimulated astrocytes to release the gliotransmitter adenosine triphosphate (ATP). ATP activation of P2X receptors excited presynaptic GABA neurons to increase GABA release, which was excitatory to the VP neurons. This trans-neuronal–glial retrograde circuit activated by ghrelin provides an alternative means of stimulation of VP release and represents a novel mechanism of neuronal control by local neuronal–glial circuits. It also provides a potential cellular mechanism for the physiological integration of energy and fluid homeostasis. PMID:24790191

EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

PubMed

Fortner, C N; Breyer, R M; Paul, R J

2001-08-01

Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva, P.; Stoff, J.S.; Solomon, R.J.

1987-01-01

Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal glands nerves, into the venous effluent of perfused glands in parallelmore » with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. VIP was measured by radioimmunoassay. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhanced local release of neurotransmitters.« less

ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion.

PubMed

Cater, Michael A; La Fontaine, Sharon; Shield, Kristy; Deal, Yolanda; Mercer, Julian F B

2006-02-01

The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway.

PubMed

Montaño, Luis M; Carbajal, Verónica; Vargas, Mario H; García-Hernández, Luz M; Díaz-Hernández, Verónica; Checa, Marco; Barajas-López, Carlos

2013-08-01

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.

A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

PubMed

Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

2018-02-05

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP.

PubMed Central

Sokolov, V S; Apell, H J; Corrie, J E; Trentham, D R

1998-01-01

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C. PMID:9591656

Transient early neurotrophin release and delayed inflammatory cytokine release by microglia in response to PAR-2 stimulation

PubMed Central

2012-01-01

Activated microglia exerts both beneficial and deleterious effects on neurons, but the signaling mechanism controlling these distinct responses remain unclear. We demonstrated that treatment of microglial cultures with the PAR-2 agonist, 2-Furoyl-LIGRLO-NH2, evoked early transient release of BDNF, while sustained PAR-2 stimulation evoked the delayed release of inflammatory cytokines (IL-1β and TNF-α) and nitric oxide. Culture medium harvested during the early phase (at 1 h) of microglial activation induced by 2-Furoyl-LIGRLO-NH2 (microglial conditioned medium, MCM) had no deleterious effects on cultured neurons, while MCM harvested during the late phase (at 72 h) promoted DNA fragmentation and apoptosis as indicated by TUNEL and annexin/PI staining. Blockade of PAR-1 during the early phase of PAR-2 stimulation enhanced BDNF release (by 11%, small but significant) while a PAR-1 agonist added during the late phase (24 h after 2-Furoyl-LIGRLO-NH2 addition) suppressed the release of cytokines and NO. The neuroprotective and neurotoxic effects of activated microglial exhibit distinct temporal profiles that are regulated by PAR-1 and PAR-2 stimulation. It may be possible to facilitate neuronal recovery and repair by appropriately timed stimulation and inhibition of microglial PAR-1 and PAR-2 receptors. PMID:22731117

Transient early neurotrophin release and delayed inflammatory cytokine release by microglia in response to PAR-2 stimulation.

PubMed

Chen, Chen-Wen; Chen, Qian-Bo; Ouyang, Qing; Sun, Ji-Hu; Liu, Fang-Ting; Song, Dian-Wen; Yuan, Hong-Bin

2012-06-25

Activated microglia exerts both beneficial and deleterious effects on neurons, but the signaling mechanism controlling these distinct responses remain unclear. We demonstrated that treatment of microglial cultures with the PAR-2 agonist, 2-Furoyl-LIGRLO-NH2, evoked early transient release of BDNF, while sustained PAR-2 stimulation evoked the delayed release of inflammatory cytokines (IL-1 β and TNF-α) and nitric oxide. Culture medium harvested during the early phase (at 1 h) of microglial activation induced by 2-Furoyl-LIGRLO-NH2 (microglial conditioned medium, MCM) had no deleterious effects on cultured neurons, while MCM harvested during the late phase (at 72 h) promoted DNA fragmentation and apoptosis as indicated by TUNEL and annexin/PI staining. Blockade of PAR-1 during the early phase of PAR-2 stimulation enhanced BDNF release (by 11%, small but significant) while a PAR-1 agonist added during the late phase (24 h after 2-Furoyl-LIGRLO-NH2 addition) suppressed the release of cytokines and NO. The neuroprotective and neurotoxic effects of activated microglial exhibit distinct temporal profiles that are regulated by PAR-1 and PAR-2 stimulation. It may be possible to facilitate neuronal recovery and repair by appropriately timed stimulation and inhibition of microglial PAR-1 and PAR-2 receptors.

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

PubMed Central

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

PubMed

Burnett, Lindsey A; Light, Mallory M; Mehrotra, Pavni; Nowak, Romana A

2012-12-01

Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine

PubMed Central

Laver, Derek R; Lenz, Gerlinde K E; Lamb, Graham D

2001-01-01

Nucleotide activation of skeletal muscle ryanodine receptors (RyRs) was studied in planar lipid bilayers in order to understand RyR regulation in vivo under normal and fatigued conditions. With ‘resting’ calcium (100 nm cytoplasmic and 1 mm luminal), RyRs had an open probability (Po) of ∼0.01 in the absence of nucleotides and magnesium. ATP reversibly activated RyRs with Po at saturation (Pmax) ∼0.33 and Ka (concentration for half-maximal activation) ∼0.36 mm and with a Hill coefficient (nH) of ∼1.8 in RyRs when Pmax < 0.5 and ∼4 when Pmax > 0.5. AMP was a much weaker agonist (Pmax∼0.09) and adenosine was weaker still (Pmax∼0.01–0.02), whereas inosine monophosphate (IMP), the normal metabolic end product of ATP hydrolysis, produced no activation at all. Adenosine acted as a competitive antagonist that reversibly inhibited ATP- and AMP-activated RyRs with nH∼1 and Ki∼0.06 mm at [ATP] < 0.5 mm, increasing 4-fold for each 2-fold increase in [ATP] above 0.5 mm. This is explained by the binding of a single adenosine preventing the cooperative binding of two ATP or AMP molecules, with dissociation constants of 0.4, 0.45 and 0.06 mm for ATP, AMP and adenosine, respectively. Importantly, IMP (≤ 8 mm) had no inhibitory effect whatsoever on ATP-activated RyRs. Mean open (τo) and closed (τc) dwell-times were more closely related to Po than to the nucleotide species or individual RyRs. At Po < 0.2, RyR regulation occurred via changes in τc, whereas at higher Po this also occurred via changes in τo. The detailed properties of activation and competitive inhibition indicated complex channel behaviour that could be explained in terms of a model involving interactions between different subunits of the RyR homotetramer. The results also show how deleterious adenosine accumulation is to the function of RyRs in skeletal muscle and, by comparison with voltage sensor-controlled Ca2+ release, indicate that voltage sensor activation requires ATP binding to

Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

PubMed

Lee, Joonsup; Wen, Beryl; Carter, Elizabeth A; Combes, Valery; Grau, Georges E R; Lay, Peter A

2017-07-01

Microvesicles (MVs) are involved in cell-cell interactions, including disease pathogenesis. Nondestructive Fourier-transform infrared (FTIR) spectra from MVs were assessed as a technique to provide new biochemical insights into a LPS-induced monocyte model of septic shock. FTIR spectroscopy provided a quick method to investigate relative differences in biomolecular content of different MV populations that was complementary to traditional semiquantitative omics approaches, with which it is difficult to provide information on relative changes between classes (proteins, lipids, nucleic acids, carbohydrates) or protein conformations. Time-dependent changes were detected in biomolecular contents of MVs and in the monocytes from which they were released. Differences in phosphatidylcholine and phosphatidylserine contents were observed in MVs released under stimulation, and higher relative concentrations of RNA and α-helical structured proteins were present in stimulated MVs compared with MVs from resting cells. FTIR spectra of stimulated monocytes displayed changes that were consistent with those observed in the corresponding MVs they released. LPS-stimulated monocytes had reduced concentrations of nucleic acids, α-helical structured proteins, and phosphatidylcholine compared with resting monocytes but had an increase in total lipids. FTIR spectra of MV biomolecular content will be important in shedding new light on the mechanisms of MVs and the different roles they play in physiology and disease pathogenesis.-Lee, J., Wen, B., Carter, E. A., Combes, V., Grau, G. E. R., Lay, P. A. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation. © FASEB.

Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells

PubMed Central

Høy, Marianne; Olsen, Hervør L; Bokvist, Krister; Buschard, Karsten; Barg, Sebastian; Rorsman, Patrik; Gromada, Jesper

2000-01-01

Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 μm and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mm) and quinine (10 μm). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 μm), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the

Magnetically stimulated ciprofloxacin release from polymeric microspheres entrapping iron oxide nanoparticles

PubMed Central

Sirivisoot, Sirinrath; Harrison, Benjamin S

2015-01-01

To extend the external control capability of drug release, iron oxide nanoparticles (NPs) encapsulated into polymeric microspheres were used as magnetic media to stimulate drug release using an alternating magnetic field. Chemically synthesized iron oxide NPs, maghemite or hematite, and the antibiotic ciprofloxacin were encapsulated together within polycaprolactone microspheres. The polycaprolactone microspheres entrapping ciprofloxacin and magnetic NPs could be triggered for immediate drug release by magnetic stimulation at a maximum value of 40%. Moreover, the microspheres were cytocompatible with fibroblasts in vitro with a cell viability percentage of more than 100% relative to a nontreated control after 24 hours of culture. Macrophage cell cultures showed no signs of increased inflammatory responses after in vitro incubation for 56 hours. Treatment of Staphylococcus aureus with the magnetic microspheres under an alternating (isolating) magnetic field increased bacterial inhibition further after 2 days and 5 days in a broth inhibition assay. The findings of the present study indicate that iron oxide NPs, maghemite and hematite, can be used as media for stimulation by an external magnetic energy to activate immediate drug release. PMID:26185446

Stimulation of GPR30 Increases Release of EMMPRIN-Containing Microvesicles in Human Uterine Epithelial Cells

PubMed Central

Light, Mallory M.; Mehrotra, Pavni; Nowak, Romana A.

2012-01-01

Context: Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. Objective: The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). Design: We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. Results: We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. Conclusions: These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology. PMID:23012390

Shock Wave Treatment Enhances Cell Proliferation and Improves Wound Healing by ATP Release-coupled Extracellular Signal-regulated Kinase (ERK) Activation*

PubMed Central

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

2014-01-01

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. PMID:25118288

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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A novel approach to regulate cell membrane permeability for ATP and NADH formation in Saccharomyces cerevisiae induced by air cold plasma

NASA Astrophysics Data System (ADS)

Dong, Xiaoyu; Liu, Tingting; Xiong, Yuqin

2017-02-01

Air cold plasma has been used as a novel method for enhancing microbial fermentation. The aim of this work was to explore the effect of plasma on membrane permeability and the formation of ATP and NADH in Saccharomyces cerevisiae, so as to provide valuable information for large-scale application of plasma in the fermentation industry. Suspensions of S. cerevisiae cells were exposed to air cold plasma for 0, 1, 2, 3, 4 and 5 min, and then subjected to various analyses prior to fermentation (0 h) and at the 9 and 21 h stages of fermentation. Compared with non-exposed cells, cells exposed to plasma for 1 min exhibited a marked increase in cytoplasmic free Ca2+ concentration as a result of the significant increase in membrane potential prior to fermentation. At the same time, the ATP level in the cell suspension decreased by about 40%, resulting in a reduction of about 60% in NADH prior to culturing. However, the levels of ATP and NADH in the culture at the 9 and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indicated that exposure of S. cerevisiae to air cold plasma could increase its cytoplasmic free Ca2+ concentration by improving the cell membrane potential, consequently leading to changes in ATP and NADH levels. Supported by National Natural Science Foundation of China (Nos. 21246012, 21306015 and 21476032).

Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine.

PubMed

Laver, D R; Lenz, G K; Lamb, G D

2001-12-15

RyRs in skeletal muscle and, by comparison with voltage sensor-controlled Ca(2+) release, indicate that voltage sensor activation requires ATP binding to the RyR to be effective.

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5-trisphosphate in cultured vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, J.B.; Smith, L.; Higgins, B.L.

1985-11-25

Inositol 1,4,5-trisphosphate (IP3) rapidly increased UVCaS efflux from a nonmitochondrial organelle in cultured vascular smooth muscle cells that were permeabilized with saponin. A nucleotide, preferably ATP, was essential for IP3-evoked UVCaS release. Two nonhydrolyzable ATP analogues satisfied the nucleotide requirement for IP3-evoked UVCaS release. IP3 strongly stimulated UVCaS efflux at low temperatures (1 to 15 degrees C). Decreasing the temperature from 37 to 4 degrees C inhibited the rate of IP3-stimulated efflux by only about 33%. The failure of such low temperatures to strongly inhibit IP3-induced UVCaS efflux suggests that IP3 activated a CaS channel, rather than a carrier, bymore » a ligand-binding, rather than a metabolic, reaction.« less

Familiar companions diminish cocaine conditioning and attenuate cocaine-stimulated dopamine release in the nucleus accumbens.

PubMed

Tzeng, Wen-Yu; Cherng, Chian-Fang G; Wang, Shyi-Wu; Yu, Lung

2016-06-01

This study aimed to assess the impact of companions on the rewarding effects of cocaine. Three cage mates, serving as companions, were housed with each experimental mouse throughout cocaine-place conditioning in a cocaine-induced conditioned place preference (CPP) paradigm using conditioning doses of 10 and 20mg/kg. The presence of companions decreased the magnitude of the CPP. At 20mg/kg, cocaine stimulated dopamine (DA) release in the nucleus accumbens as evidenced by a significant decrease in total (spontaneous and electrical stimulation-provoked) DA release in accumbal superfusate samples. The presence of companions prevented this cocaine-stimulated DA release; such a reduction in cocaine-induced DA release may account for the reduction in the magnitude of the CPP in the presence of the companions. Furthermore, cocaine pretreatment (2.5mg/kg) was found to prevent the companion-produced decreases in cocaine (10mg/kg/conditioning)-induced CPP as well as the cocaine (10mg/kg)-stimulated DA release. Moreover, the presence of methamphetamine (MA) (1mg/kg)-treated companions decreased cocaine (20mg/kg/conditioning)-induced CPP and prevented the cocaine (20mg/kg)-stimulated DA release. Finally, the presence of companions decreased the magnitude of the CPP could not seem to be accounted for by cocaine-stimulated corticosterone (CORT) release. Taken together, these results indicate that familiar companions, regardless of their pharmacological status, may exert dampening effects on CPP induced by moderate to high conditioning doses of cocaine, at least in part, by preventing cocaine-stimulated DA release in the nucleus accumbens. Copyright © 2016 Elsevier B.V. All rights reserved.

Copper stimulation of LHRH release from median eminence explants. III. A process dependent on extracellular sodium.

PubMed

Colombani-Vidal, M; Barnea, A

1986-01-01

Copper, complexed to histidine (CuHis), stimulates LHRH release from explants of the median eminence area (MEA). To gain further understanding of the mechanism of copper action, in this study, we assessed the Na+ and energy requirements for CuHis stimulation of LHRH release. MEA explants, obtained from adult male rats, were incubated at 37 degrees C for 15 min with 100 microM CuHis and then for 45 min in CuHis-free medium (Krebs-Ringer-phosphate buffer, pH 7.4). LHRH released into the medium was evaluated by RIA. When the incubation buffer contained 143 mM Na+, CuHis stimulated the release of LHRH from a basal level of 17.2 +/- 1.26 (mean +/- SEM, n = 7) to 74.5 +/- 6.2 pg/60 min per MEA. When [Na+] was reduced to 16 mM Na+ (by substituting with Li+), CuHis-stimulated LHRH release was inhibited by 80% (p less than 0.001); indicating a requirement for Na+. In addition, we found that CuHis-stimulated LHRH release was a saturable function of Na+ concentration; saturation achieved with about 100 mM Na+. To assess the requirement for Na+ transport, we evaluated the effect of 1 mM ouabain, 10 microM tetrodotoxin (TTX), or 100 microM amiloride on CuHis stimulation of LHRH release. Ouabain inhibited CuHis stimulation of LHRH release by 80%, whereas TTX and amiloride were ineffective. In addition, we observed that CuHis did not stimulate LHRH release when incubation was carried out at 4 degrees C or at 37 degrees C in the presence of 5 mM KCN.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugal air-assisted melt agglomeration for fast-release "granulet" design.

PubMed

Wong, Tin Wui; Musa, Nafisah

2012-07-01

Conventional melt pelletization and granulation processes produce round and dense, and irregularly shaped but porous agglomerates respectively. This study aimed to design centrifugal air-assisted melt agglomeration technology for manufacture of spherical and yet porous "granulets" for ease of downstream manufacturing and enhancing drug release. A bladeless agglomerator, which utilized shear-free air stream to mass the powder mixture of lactose filler, polyethylene glycol binder and poorly water-soluble tolbutamide drug into "granulets", was developed. The inclination angle and number of vane, air-impermeable surface area of air guide, processing temperature, binder content and molecular weight were investigated with reference to "granulet" size, shape, texture and drug release properties. Unlike fluid-bed melt agglomeration with vertical processing air flow, the air stream in the present technology moved centrifugally to roll the processing mass into spherical but porous "granulets" with a drug release propensity higher than physical powder mixture, unprocessed drug and dense pellets prepared using high shear mixer. The fast-release attribute of "granulets" was ascribed to porous matrix formed with a high level of polyethylene glycol as solubilizer. The agglomeration and drug release outcomes of centrifugal air-assisted technology are unmet by the existing high shear and fluid-bed melt agglomeration techniques. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional studies of ATP sulfurylase from Penicillium chrysogenum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seubert, P.A.

1985-01-01

ATP sulfurylase from Penicillium chrysogenum has a specific activity (V/sub max/) of 6-7 units x mg protein/sup -1/ determined with the physiological substrates of MgATP and SO/sub 4//sup 2 -/ and assayed by (A) initial velocity measurements with APS kinase and inorganic pyrophosphatase present and (B) analysis of nonlinear reaction progress curves. The fact both assays give the same results show the intrinsic activity of ATP sulfurylase is much higher than previously reported. In initial velocity dead-end inhibition studies, the sulfate analog S/sub 2/O/sub 3//sup 2 -/ is a competitive inhibitor of SO/sub 42/..sqrt.. and a noncompetitive inhibitor of MgATP.more » Monovalent oxyanions such as NO/sub 3//sup -/, ClO/sub 3//sup -/, ClO/sub 4//sup -/, and FSO/sub 3//sup -/ behave as uncompetitive inhibitors of MgATP and thus seem not to be true sulfate analogs. The reverse reaction was assayed by the pyrophosphate dependent release of /sup 35/SO/sub 4//sup 2 -/ from AP/sup 35/S. Product inhibition by MgATP or SO/sub 4//sup 2 -/ is competitive with APS and mixed-type with PP/sub i/. Imidodiphosphate can serve as an alternative substrate for PP/sub i/. ATP sulfurylase binds (but does not hydrolyze) APS. A Scatchard plot of the APS binding is nonlinear, suggesting at least two types of sites. The cumulative results are qualitatively consistent with the random addition of MgATP and SO/sub 4//sup 2 -/ and the ordered release of first MgPP/sub i/ then APS, with APS release being partially rate limiting. Certain quantitative discrepancies suggest either an unknown variable (e.g. enzyme concentration) complicates the analysis or, in light of binding studies that the actual mechanism is more complicated (e.g. alternating sites) than any of the conventional models examined.« less

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

PubMed

Sunggip, Caroline; Shimoda, Kakeru; Oda, Sayaka; Tanaka, Tomohiro; Nishiyama, Kazuhiro; Mangmool, Supachoke; Nishimura, Akiyuki; Numaga-Tomita, Takuro; Nishida, Motohiro

2018-01-01

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca 2+ -dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca 2+ -mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca 2+ signaling requires inositol 1,4,5-trisphosphate (IP 3 ) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca 2+ /NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP 3 -mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Communication between the N and C Termini Is Required for Copper-stimulated Ser/Thr Phosphorylation of Cu(I)-ATPase (ATP7B)*

PubMed Central

Braiterman, Lelita T.; Gupta, Arnab; Chaerkady, Raghothama; Cole, Robert N.; Hubbard, Ann L.

2015-01-01

The Wilson disease protein ATP7B exhibits copper-dependent trafficking. In high copper, ATP7B exits the trans-Golgi network and moves to the apical domain of hepatocytes where it facilitates elimination of excess copper through the bile. Copper levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low copper and becomes more phosphorylated (“hyperphosphorylated”) in elevated copper. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the trans-Golgi network. We performed comprehensive phosphoproteomics of ATP7B in low versus high copper, which revealed that 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of these were copper-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all six N-terminal metal binding domains did not block copper-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that nine of 15 Ser/Thr residues in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that copper binding at the N terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon copper-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed that copper-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune copper sequestration. PMID:25666620

Release mechanism of high mobility group nucleosome binding domain 1 from lipopolysaccharide-stimulated macrophages.

PubMed

Murakami, Taisuke; Hu, Zhongshuang; Tamura, Hiroshi; Nagaoka, Isao

2016-04-01

Alarmins are identified as endogenous mediators that have potent immune-activating abilities. High mobility group nucleosome binding domain 1 (HMGN1), a highly conserved, non-histone chromosomal protein, which binds to the inner side of the nucleosomal DNA, regulates chromatin dynamics and transcription in cells. Furthermore, HMGN1 acts as a cytokine in the extracellular milieu by inducing the recruitment and maturation of antigen-presenting cells (dendritic cells) to enhance Th1-type antigen-specific immune responses. Thus, HMGN1 is expected to act as an alarmin, when released into the extracellular milieu. The present study investigated the release mechanism of HMGN1 from macrophages using mouse macrophage‑like RAW264.7 cells. The results indicated that HMGN1 was released from lipopolysaccharide (LPS)‑stimulated RAW264.7 cells, accompanied by cell death as assessed by the release of lactate dehydrogenase (LDH). Subsequently, the patterns of cell death involved in HMGN1 release from LPS‑stimulated RAW264.7 cells were determined using a caspase‑1 inhibitor, YVAD, and a necroptosis inhibitor, Nec‑1. YVAD and Nec‑1 did not alter LPS‑induced HMGN1 and LDH release, suggesting that pyroptosis (caspase‑1‑activated cell death) and necroptosis are not involved in the release of HMGN1 from LPS‑stimulated RAW264.7 cells. In addition, flow cytometric analysis indicated that LPS stimulation did not induce apoptosis but substantially augmented necrosis, as evidenced by staining with annexin V/propidium iodide. Together these findings suggest that HMGN1 is extracellularly released from LPS‑stimulated RAW264.7 macrophage‑like cells, accompanied by unprogrammed necrotic cell death but not pyroptosis, necroptosis or apoptosis.

Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways

NASA Technical Reports Server (NTRS)

McAllister, T. N.; Frangos, J. A.

1999-01-01

Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.

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

PubMed Central

Crecelius, Anne R.; Kirby, Brett S.; Richards, Jennifer C.

2013-01-01

Intravascular adenosine triphosphate (ATP) evokes vasodilation and is implicated in the regulation of skeletal muscle blood flow during exercise. Mechanical stresses to erythrocytes and endothelial cells stimulate ATP release in vitro. How mechanical effects of muscle contractions contribute to increased plasma ATP during exercise is largely unexplored. We tested the hypothesis that simulated mechanical effects of muscle contractions increase [ATP]venous and ATP effluent in vivo, independent of changes in tissue metabolic demand, and further increase plasma ATP when superimposed with mild-intensity exercise. In young healthy adults, we measured forearm blood flow (FBF) (Doppler ultrasound) and plasma [ATP]v (luciferin-luciferase assay), then calculated forearm ATP effluent (FBF×[ATP]v) during rhythmic forearm compressions (RFC) via a blood pressure cuff at three graded pressures (50, 100, and 200 mmHg; Protocol 1; n = 10) and during RFC at 100 mmHg, 5% maximal voluntary contraction rhythmic handgrip exercise (RHG), and combined RFC + RHG (Protocol 2; n = 10). [ATP]v increased from rest with each cuff pressure (range 144–161 vs. 64 ± 13 nmol/l), and ATP effluent was graded with pressure. In Protocol 2, [ATP]v increased in each condition compared with rest (RFC: 123 ± 33; RHG: 51 ± 9; RFC + RHG: 96 ± 23 vs. Mean Rest: 42 ± 4 nmol/l; P < 0.05), and ATP effluent was greatest with RFC + RHG (RFC: 5.3 ± 1.4; RHG: 5.3 ± 1.1; RFC + RHG: 11.6 ± 2.7 vs. Mean Rest: 1.2 ± 0.1 nmol/min; P < 0.05). We conclude that the mechanical effects of muscle contraction can 1) independently elevate intravascular ATP draining quiescent skeletal muscle without changes in local metabolism and 2) further augment intravascular ATP during mild exercise associated with increases in metabolism and local deoxygenation; therefore, it is likely one stimulus for increasing intravascular ATP during exercise in humans. PMID:23429876

Copper amplification of prostaglandin E/sub 2/ stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnea, A.; Cho, G.

1987-01-01

The authors have shown that copper amplifies prostaglandin E/sub 2/ (PGE/sub 2/) stimulation of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA) and that this process is calcium-dependent. Since a Ca-cAMP pathway has been implicated in PGE/sub 2/ action on the LH-RH neuron, in this study the authors wished to ascertain if copper exerts its effect on the PGE/sub 2/ receptor or on a postreceptor component involved in PGE/sub 2/ action. MEA of adult male rats were incubated for 5 min with 200 ..mu..M Cu/histidine and then incubated for 15 min either with 10 ..mu..M PGE/submore » 2/ (Cu/PGE/sub 2/), 100 ..mu..M forskolin (Cu/forskolin), or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Cu/cAMP). Basal release of LH-RH was 4.6 +/- 0.45 pg/15 min per MEA determined by radioimmunoassay. Net stimulated release during the 15-min exposure to PGE/sub 2/, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate was 3.6 +/- 0.52, 3.1 +/- 0.39, and 1.6 +/- 0.42 pg/15 min per MEA, respectively. Net stimulated release after exposure to Cu/PGE/sub 2/, Cu/forskolin, or Cu/cAMP indicated that copper amplifies the action of PGE/sub 2/ and forskolin but not cAMP action. When MEA were exposed to a mixture of PGE/sub 2/ and forskolin for 15 min, the effects of these two secretagogues on LH-RH release were not additive. In contrast to PGE/sub 2/ and forskolin, copper did not amplify K/sup +/ stimulation of OH-RH release. These results are supportive of the proposition that PGE/sub 2/ stimulation of OH-RH release is mediated by the Ca-cAMP pathway and that copper amplification of PGE/sub 2/ action is a postreceptor event.« less

Fructose stimulates GLP-1 but not GIP secretion in mice, rats, and humans

PubMed Central

Kuhre, Rune E.; Gribble, Fiona M.; Hartmann, Bolette; Reimann, Frank; Windeløv, Johanne A.; Rehfeld, Jens F.

2014-01-01

Nutrients often stimulate gut hormone secretion, but the effects of fructose are incompletely understood. We studied the effects of fructose on a number of gut hormones with particular focus on glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In healthy humans, fructose intake caused a rise in blood glucose and plasma insulin and GLP-1, albeit to a lower degree than isocaloric glucose. Cholecystokinin secretion was stimulated similarly by both carbohydrates, but neither peptide YY3–36 nor glucagon secretion was affected by either treatment. Remarkably, while glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, whereas only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L cells, fructose was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by ATP-sensitive potassium channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might be useful targets for type 2 diabetes mellitus and obesity drug development. PMID:24525020

Self-soothing behaviors with particular reference to oxytocin release induced by non-noxious sensory stimulation.

PubMed

Uvnäs-Moberg, Kerstin; Handlin, Linda; Petersson, Maria

2014-01-01

Oxytocin, a hypothalamic nonapeptide, is linked to increased levels of social interaction, well-being and anti-stress effects. The effects of oxytocin that is released by sensory stimulation during different kinds of interactive behaviors are often underestimated or even forgotten. In fact, many of the positive effects caused during interaction, such a wellbeing, stress reduction and even health promotion, are indeed linked to oxytocin released in response to activation of various types of sensory nerves. Oxytocin is released in response to activation of sensory nerves during labor, breastfeeding and sexual activity. In addition oxytocin is released in response to low intensity stimulation of the skin, e.g., in response to touch, stroking, warm temperature, etc. Consequently oxytocin is not only released during interaction between mothers and infants, but also during positive interaction between adults or between humans and animals. Finally oxytocin is also released in response to suckling and food intake. Oxytocin released in the brain in response to sensory stimulation as a consequence of these types of interactive behaviors, contributes to every day wellbeing and ability to handle stress. Food intake or sex may be used or even abused to achieve oxytocin-linked wellbeing and stress relief to compensate for lack of good relationships or when the levels of anxiety are high. The present review article will summarize the role played by oxytocin released by sensory (in particular somatosensory) stimulation, during various kinds of interactive behaviors. Also the fact that the anti-stress effects of oxytocin are particularly strong when oxytocin is released in response to "low intensity" stimulation of the skin will be highlighted.

Self-soothing behaviors with particular reference to oxytocin release induced by non-noxious sensory stimulation

PubMed Central

Uvnäs-Moberg, Kerstin; Handlin, Linda; Petersson, Maria

2015-01-01

Oxytocin, a hypothalamic nonapeptide, is linked to increased levels of social interaction, well-being and anti-stress effects. The effects of oxytocin that is released by sensory stimulation during different kinds of interactive behaviors are often underestimated or even forgotten. In fact, many of the positive effects caused during interaction, such a wellbeing, stress reduction and even health promotion, are indeed linked to oxytocin released in response to activation of various types of sensory nerves. Oxytocin is released in response to activation of sensory nerves during labor, breastfeeding and sexual activity. In addition oxytocin is released in response to low intensity stimulation of the skin, e.g., in response to touch, stroking, warm temperature, etc. Consequently oxytocin is not only released during interaction between mothers and infants, but also during positive interaction between adults or between humans and animals. Finally oxytocin is also released in response to suckling and food intake. Oxytocin released in the brain in response to sensory stimulation as a consequence of these types of interactive behaviors, contributes to every day wellbeing and ability to handle stress. Food intake or sex may be used or even abused to achieve oxytocin-linked wellbeing and stress relief to compensate for lack of good relationships or when the levels of anxiety are high. The present review article will summarize the role played by oxytocin released by sensory (in particular somatosensory) stimulation, during various kinds of interactive behaviors. Also the fact that the anti-stress effects of oxytocin are particularly strong when oxytocin is released in response to “low intensity” stimulation of the skin will be highlighted. PMID:25628581

Bubble Jet agent release cartridge for chemical single cell stimulation.

PubMed

Wangler, N; Welsche, M; Blazek, M; Blessing, M; Vervliet-Scheebaum, M; Reski, R; Müller, C; Reinecke, H; Steigert, J; Roth, G; Zengerle, R; Paust, N

2013-02-01

We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d <30 μm) on-demand micro gradients can be generated for the specific manipulation of single cells. A single channel and a double channel agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15 pl and V = 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.

Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

PubMed

Gicquel, Thomas; Victoni, Tatiana; Fautrel, Alain; Robert, Sacha; Gleonnec, Florence; Guezingar, Marie; Couillin, Isabelle; Catros, Véronique; Boichot, Elisabeth; Lagente, Vincent

2014-04-01

Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury. © 2014 Wiley Publishing Asia Pty Ltd.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

PubMed Central

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

"Host tissue damage" signal ATP promotes non-directional migration and negatively regulates toll-like receptor signaling in human monocytes.

PubMed

Kaufmann, Andreas; Musset, Boris; Limberg, Sven H; Renigunta, Vijay; Sus, Rainer; Dalpke, Alexander H; Heeg, Klaus M; Robaye, Bernard; Hanley, Peter J

2005-09-16

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.

Adenosine transiently modulates stimulated dopamine release in the caudate putamen via A1 receptors

PubMed Central

Ross, Ashley E.; Venton, B. Jill

2014-01-01

Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 µM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7 %, similar to the 54 ± 6 % decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 minutes. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. PMID:25219576

Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3-CA1 synapses of adult mice are modulated by glutamate and ATP.

PubMed

Tang, Wannan; Szokol, Karolina; Jensen, Vidar; Enger, Rune; Trivedi, Chintan A; Hvalby, Øivind; Helm, P Johannes; Looger, Loren L; Sprengel, Rolf; Nagelhus, Erlend A

2015-02-18

To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) signals in astrocytic processes at CA3-CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP. Copyright © 2015 the authors 0270-6474/15/353016-06$15.00/0.

Interleukin 6 stimulates hepatic glucose release from prelabeled glycogen pools

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritchie, D.G.

1990-01-01

Cytokines, derived from a wide variety of cell types, are now believed to initiate many of the physiological responses accompanying the inflammatory phase that follows either Gram-negative septicemia or thermal injury. Because hypoglycemia (after endotoxic challenge) and hyperglycemia (after thermal injury) represent well-characterized responses to these injuries, we sought to determine whether hepatic glycogen metabolism could be altered by specific cytokines. Cultured adult rat hepatocytes were prelabeled with ({sup 14}C)glucose for 24 h, a procedure that resulted in the labeling of hepatic glycogen pools that subsequently could be depleted (with concomitant ({sup 14}C)glucose release) by either glucagon or norepinephrine. Aftermore » the addition of a highly concentrated human monocyte-conditioned medium (MCM) or various cytokines to these prelabeled cells, ({sup 14}C)glucose release was stimulated by MCM and recombinant human interleukin 6 (IL-6) but was not stimulated by other cytokines tested. Furthermore, only antisera to IL-6 were capable of reducing the glucose-releasing factor activity found in MCM. These data therefore suggest a novel glucoregulatory role for IL-6.« less

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

PubMed Central

Fagan, J M; Waxman, L; Goldberg, A L

1987-01-01

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors. Images Fig. 2. PMID:2820375

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanism of the beneficial effects of ATP-MgCl2 following trauma-hemorrhage and resuscitation: downregulation of inflammatory cytokine (TNF, IL-6) release.

PubMed

Wang, P; Ba, Z F; Morrison, M H; Ayala, A; Dean, R E; Chaudry, I H

1992-04-01

Although ATP-MgCl2 improves hepatocellular function in a nonheparinized model of trauma-hemorrhage and crystalloid resuscitation, it remains unknown whether the beneficial effects of this agent are due to downregulation of the release of the inflammatory cytokines, tumor necrosis factor (TNF), and interleukin-6 (IL-6) under those conditions. To study this, rats underwent a 5-cm laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mm Hg until 40% of maximum bleedout volume was returned in the form of Ringer's lactate (RL). The animals were then resuscitated with four times the volume of shed blood with RL over 60 min. ATP-MgCl2 (50 mumoles/kg body weight each) or an equivalent volume of normal saline was infused intravenously for 95 min. This infusion was started during the last 15 min of RL resuscitation. Plasma levels of TNF and IL-6 were measured at 1.5 hr after the completion of resuscitation by cytokine-dependent cellular assays. Hepatic blood flow was determined by in vivo indocyanine green clearance (corrected by hepatic extraction ratio for indocyanine green), radioactive microspheres, and [3H]-galactose clearance techniques. The results indicate that the levels of circulating TNF and IL-6 increased significantly in the hemorrhaged-resuscitated animals. ATP-MgCl2 treatment, however, markedly decreased the synthesis and/or release of these cytokines to levels similar to the sham group. The markedly decreased hepatic blood flow (as determined by three different methods) and hepatic extraction ratio for indocyanine green were also restored by ATP-MgCl2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel cellular bouton structure activated by ATP in the vascular wall of porcine retinal arterioles.

PubMed

Misfeldt, Mikkel Wölck; Aalkjaer, Christian; Simonsen, Ulf; Bek, Toke

2010-12-01

The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5'-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles. Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall. Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5'-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone. The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

Effectiveness of water-air and octanol-air partition coefficients to predict lipophilic flavor release behavior from O/W emulsions.

PubMed

Tamaru, Shunji; Igura, Noriyuki; Shimoda, Mitsuya

2018-01-15

Flavor release from food matrices depends on the partition of volatile flavor compounds between the food matrix and the vapor phase. Thus, we herein investigated the relationship between released flavor concentrations and three different partition coefficients, namely octanol-water, octanol-air, and water-air, which represented the oil, water, and air phases present in emulsions. Limonene, 2-methylpyrazine, nonanal, benzaldehyde, ethyl benzoate, α-terpineol, benzyl alcohol, and octanoic acid were employed. The released concentrations of these flavor compounds from oil-in-water (O/W) emulsions were measured under equilibrium using static headspace gas chromatography. The results indicated that water-air and octanol-air partition coefficients correlated with the logarithms of the released concentrations in the headspace for highly lipophilic flavor compounds. Moreover, the same tendency was observed over various oil volume ratios in the emulsions. Our findings therefore suggest that octanol-air and water-air partition coefficients can be used to predict the released concentration of lipophilic flavor compounds from O/W emulsions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a new Mpl-interacting protein, Atp5d.

PubMed

Liu, Hongyan; Zhao, Zhenhu; Zhong, Yuxu; Shan, Yajun; Sun, Xiaohong; Mao, Bingzhi; Cong, Yuwen

2014-06-01

Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

PubMed

Liu, Jun; Liu, Wenjing; Yang, Jun

2016-02-11

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

PubMed Central

Liu, Jun; Liu, Wenjing; Yang, Jun

2016-01-01

We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

Simulation of radiation energy release in air showers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glaser, Christian; Erdmann, Martin; Hörandel, Jörg R.

2016-09-01

A simulation study of the energy released by extensive air showers in the form of MHz radiation is performed using the CoREAS simulation code. We develop an efficient method to extract this radiation energy from air-shower simulations. We determine the longitudinal profile of the radiation energy release and compare it to the longitudinal profile of the energy deposit by the electromagnetic component of the air shower. We find that the radiation energy corrected for the geometric dependence of the geomagnetic emission scales quadratically with the energy in the electromagnetic component of the air shower with a second-order dependence on themore » atmospheric density at the position of the maximum shower development X {sub max}. In a measurement where X {sub max} is not accessible, this second order dependence can be approximated using the zenith angle of the incoming direction of the air shower with only a minor loss in accuracy. Our method results in an intrinsic uncertainty of 4% in the determination of the energy in the electromagnetic air-shower component, which is well below current experimental uncertainties.« less

Aptamer loaded MoS2 nanoplates as nanoprobes for detection of intracellular ATP and controllable photodynamic therapy

NASA Astrophysics Data System (ADS)

Jia, Li; Ding, Lin; Tian, Jiangwei; Bao, Lei; Hu, Yaoping; Ju, Huangxian; Yu, Jun-Sheng

2015-09-01

In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP-mediated release led to the generation of 1O2 under 660 nm laser irradiation, which could induce tumor cell death with a lysosomal pathway. The controllable PDT provided a model approach for design of multifunctional theranostic nanoprobes. These results also promoted the development and application of MoS2 nanoplate-based platforms in biomedicine.In this work we designed a MoS2 nanoplate-based nanoprobe for fluorescence imaging of intracellular ATP and photodynamic therapy (PDT) via ATP-mediated controllable release of 1O2. The nanoprobe was prepared by simply assembling a chlorine e6 (Ce6) labelled ATP aptamer on MoS2 nanoplates, which have favorable biocompatibility, unusual surface-area-to-mass ratio, strong affinity to single-stranded DNA, and can quench the fluorescence of Ce6. After the nanoprobe was internalized into the cells and entered ATP-abundant lysosomes, its recognition to ATP led to the release of the single-stranded aptamer from MoS2 nanoplates and thus recovered the fluorescence of Ce6 at an excitation wavelength of 633 nm, which produced a highly sensitive and selective method for imaging of intracellular ATP. Meanwhile, the ATP

CO2/HCO3−- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

PubMed Central

Zippin, Jonathan H.; Chen, Yanqiu; Straub, Susanne G.; Hess, Kenneth C.; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G.; Sharp, Geoffrey W. G.; Levin, Lonny R.; Buck, Jochen

2013-01-01

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F F{sub 1}-ATP synthase and ubiquinone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shertzer, Howard G.; Genter, Mary Beth; Shen, Dongxiao

2006-12-15

Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q wasmore » more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.« less

Detection of the urban release of a bacillus anthracis simulant by air sampling.

PubMed

Garza, Alexander G; Van Cuyk, Sheila M; Brown, Michael J; Omberg, Kristin M

2014-01-01

In 2005 and 2009, the Pentagon Force Protection Agency (PFPA) staged deliberate releases of a commercially available organic pesticide containing Bacillus amyloliquefaciens to evaluate PFPA's biothreat response protocols. In concert with, but independent of, these releases, the Department of Homeland Security sponsored experiments to evaluate the efficacy of commonly employed air and surface sampling techniques for detection of an aerosolized biological agent. High-volume air samplers were placed in the expected downwind plume, and samples were collected before, during, and after the releases. Environmental surface and personal air samples were collected in the vicinity of the high-volume air samplers hours after the plume had dispersed. The results indicate it is feasible to detect the release of a biological agent in an urban area both during and after the release of a biological agent using high-volume air and environmental sampling techniques.

Anaplerotic input is sufficient to induce time-dependent potentiation of insulin release in rat pancreatic islets.

PubMed

Gunawardana, Subhadra C; Liu, Yi-Jia; Macdonald, Michael J; Straub, Susanne G; Sharp, Geoffrey W G

2004-11-01

Nutrients that induce biphasic insulin release, such as glucose and leucine, provide acetyl-CoA and anaplerotic input in the beta-cell. The first phase of release requires increased ATP production leading to increased intracellular Ca(2+) concentration ([Ca(2+)](i)). The second phase requires increased [Ca(2+)](i) and anaplerosis. There is strong evidence to indicate that the second phase is due to augmentation of Ca(2+)-stimulated release via the K(ATP) channel-independent pathway. To test whether the phenomenon of time-dependent potentiation (TDP) has similar properties to the ATP-sensitive K(+) channel-independent pathway, we monitored the ability of different agents that provide acetyl-CoA and anaplerotic input or both of these inputs to induce TDP. The results show that anaplerotic input is sufficient to induce TDP. Interestingly, among the agents tested, the nonsecretagogue glutamine, the nonhydrolyzable analog of leucine aminobicyclo[2.2.1]heptane-2-carboxylic acid, and succinic acid methyl ester all induced TDP, and all significantly increased alpha-ketoglutarate levels in the islets. In conclusion, anaplerosis that enhances the supply and utilization of alpha-ketoglutarate in the tricarboxylic acid cycle appears to play an essential role in the generation of TDP.

Release of “neurokinin” on nervous and electrical stimulation of a frog stomach muscle preparation

PubMed Central

Singh, I.

1964-01-01

Activation of a frog stomach muscle preparation by electrical stimulation of a vagus nerve or by direct stimulation released two polypeptides. One was destroyed by trypsin or chymotrypsin in about 10 min; the activity of the other was enhanced by trypsin for about 10 min, but was destroyed by chymotrypsin. Similar stimulation of dog stomach muscle did not release these polypeptides. Correspondingly, the transmission from vagus nerve to stomach muscle in the frog was resistant to atropine, but was blocked by atropine in the dog. PMID:14190475

Minoxidil opens mitochondrial K(ATP) channels and confers cardioprotection.

PubMed

Sato, Toshiaki; Li, Yulong; Saito, Tomoaki; Nakaya, Haruaki

2004-01-01

1. ATP-sensitive potassium channel in the mitochondrial inner membrane (mitoK(ATP) channel) rather than in the sarcolemma (sarcK(ATP) channel) appears to play an important role in cardioprotection. We examined the effect of minoxidil, a potent antihypertensive agent and hair growth stimulator, on sarcK(ATP) and mitoK(ATP) channels in guinea-pig ventricular myocytes. 2. Minoxidil activated a glybenclamide-sensitive sarcK(ATP) channel current in the whole-cell recording mode with an EC(50) of 182.6 microm. Minoxidil reversibly increased the flavoprotein oxidation, an index of mitoK(ATP) channel activity, in a concentration-dependent manner. The EC(50) for mitoK(ATP) channel activation was estimated to be 7.3 microm; this value was notably approximately 25-fold lower than that for sarcK(ATP) channel activation. 3. Minoxidil (10 microm) significantly attenuated the ouabain-induced increase of mitochondrial Ca(2+) concentration, which was measured by loading cells with rhod-2 fluorescence. Furthermore, pretreatment with minoxidil (10 microm) before 20-min no-flow ischaemia significantly improved the recovery of developed tension measured after 60 min of reperfusion in coronary perfused guinea-pig ventricular muscles. These cardioprotective effects of minoxidil were completely abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microm). 4. Our results indicate that minoxidil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by the selective activation of mitoK(ATP) channels.

Identification of P2X3 and P2X7 Purinergic Receptors Activated by ATP in Rat Lacrimal Gland

PubMed Central

Vrouvlianis, Joanna; Scott, Rachel; Dartt, Darlene A.

2011-01-01

Purpose. To identify the type of purinergic receptors activated by adenosine triphosphate (ATP) in rat lacrimal gland and to determine their role in protein secretion. Methods. Purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Acini from rat lacrimal gland were isolated by collagenase digestion. Acini were incubated with the fluorescence indicator fura-2 tetra-acetoxylmethyl ester, and intracellular [Ca2+] ([Ca2+]i) was determined. Protein secretion was measured by fluorescence assay. Results. The authors previously showed that P2X7 receptors were functional in the lacrimal gland. In this study, they show that P2X1–4, and P2X6receptors were identified in the lacrimal gland by RT-PCR, Western blot, and immunofluorescence analyses. P2X5 receptors were not detected. ATP increased [Ca2+]i and protein secretion in a concentration-dependent manner. Removal of extracellular Ca2+ significantly reduced the ATP-stimulated increase in [Ca2+]i. Repeated applications of ATP caused desensitization of the [Ca2+]i response. Incubation with the P2X1 receptor inhibitor NF023 did not alter ATP-stimulated [Ca2+]i. Incubation with zinc, which potentiates P2X2 and P2X4 receptor responses, or lowering the pH to 6.8, which potentiates P2X2 receptor responses, did not alter the ATP-stimulated [Ca2+]i. P2X3 receptor inhibitors A-317491 and TNP-ATP significantly decreased ATP-stimulated [Ca2+]i and protein secretion, whereas the P2X3 receptor agonist α,β methylene ATP significantly increased them. The P2X7 receptor inhibitor A438079 had no effect on ATP-stimulated [Ca2+]i at 10−6 M but did have an effect at 10−4 M. Conclusions. Purinergic receptors P2X1–4 and P2X6 are present in the lacrimal gland. ATP uses P2X3 and P2X7 receptors to stimulate an increase in [Ca2+]i and protein secretion. PMID:21421865

Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter

PubMed Central

Lu, Shuo; Zgurskaya, Helen I.

2012-01-01

Summary MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both gram-positive and gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex. PMID:23057817

Ventilation-induced release of phosphatidylcholine from neonatal-rat lungs in vitro.

PubMed Central

Nijjar, M S

1984-01-01

Factors regulating the release of phosphatidylcholine (PC) from neonatal-rat lungs were investigated. The results show that the release of prelabelled PC from the newborn-rat lung was augmented by air ventilation at the onset of breathing. This response was mimicked in lungs of pups delivered 1 day before term and allowed to breathe for different time intervals. Anoxia further augmented the ventilation-enhanced PC release from the newborn-rat lungs. The ventilation-induced release of PC was not abolished by the prior treatment of pups in utero or mothers in vivo with phenoxybenzamine, propranolol or atropine, suggesting the lack of receptor stimulation in the ventilation-enhanced PC release at birth. The results also show that ventilation stimulated [methyl-14C]choline incorporation into lung PC, presumably to replenish the depleted surfactant stores. The ratio of adenylate cyclase/cyclic AMP phosphodiesterase activities, which reflects cyclic AMP levels in the developing rat lungs, did not change during the 120 min of air ventilation when the release of PC was much enhanced, implying that cyclic AMP may not be involved. This confirms our conclusion that stimulation of beta-adrenergic receptor was not involved in the air-ventilation-enhanced release of PC. Since the cell number or size did not change during 120 min of ventilation when the alveolar-cell surface was maximally distended, it is suggested that distension of alveolar wall by air ventilation at the onset of breathing may bring the lamellar bodies containing surfactant close to the luminal surface of alveolar type II cells, thereby enhancing their fusion and extrusion by exocytosis. PMID:6477485

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein*

PubMed Central

Ah-Seng, Yoan; Lopez, Frederic; Pasta, Franck; Lane, David; Bouet, Jean-Yves

2009-01-01

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis. PMID:19740757

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. © 2015 Authors; published by Portland Press Limited.

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

PubMed Central

Nguyen, Michael D.; Venton, B. Jill

2014-01-01

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

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

PubMed

Nguyen, Michael D; Venton, B Jill

2015-01-01

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

Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.

PubMed

Haas, Brigitte; Schipke, Carola G; Peters, Oliver; Söhl, Goran; Willecke, Klaus; Kettenmann, Helmut

2006-02-01

In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from 'sniffer cells', a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the 'sniffer cell' population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.

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.

ATP as a biomarker of viable microorganisms in clean-room facilities.

PubMed

Venkateswaran, Kasthuri; Hattori, Noriaki; La Duc, Myron T; Kern, Roger

2003-03-01

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.

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.

Sustained release carrier for adenosine triphosphate as signaling molecule.

PubMed

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

2014-12-10

Adenosine triphosphate (ATP) is a molecule with a fascinating variety of intracellular and extracellular biological functions that go far beyond energy metabolism. Due to its limited passive diffusion through biological membranes, controlled release systems may allow to interact with ATP-mediated extracellular processes. In this study, two release systems were explored to evaluate the capacity for either long-term or short-term release: (i) Poly[(rac-lactide)-co-glycolide] (PLGA) implant rods were capable of ATP release over days to weeks, depending on the PLGA molecular weight and end-group capping, but were also associated with partial hydrolytic degradation of ATP to ADP and AMP, but not adenosine. (ii) Thermosensitive methylcellulose hydrogels with a gelation occurring at body temperature allowed combining adjustable loading levels and the capacity for injection, with injection forces less than 50N even for small 27G needles. Finally, a first in vitro study illustrated purinergic-triggered response of primary murine microglia to ATP released from hydrogels, demonstrating the potential relevance for biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

A New Slow Releasing, H2S Generating Compound, GYY4137 Relaxes Spontaneous and Oxytocin-Stimulated Contractions of Human and Rat Pregnant Myometrium

PubMed Central

Robinson, Hayley; Wray, Susan

2012-01-01

Better tocolytics are required to help prevent preterm labour. The gaseotransmitter Hydrogen sulphide (H2S) has been shown to reduce myometrial contractility and thus is of potential interest. However previous studies used NaHS, which is toxic and releases H2S as a non-physiological bolus and thus alternative H2S donors are sought. GYY4137 has been developed to slowly release H2S and hence better reflect endogenous physiological release. We have examined its effects on spontaneous and oxytocin-stimulated contractility and compared them to NaHS, in human and rat myometrium, throughout gestation. The effects on contractility in response to GYY4137 (1 nM–1 mM) and NaHS (1 mM) were examined on myometrial strips from, biopsies of women undergoing elective caesarean section or hysterectomy, and from non-pregnant, 14, 18, 22 day (term) gestation or labouring rats. In pregnant rat and human myometrium dose-dependent and significant decreases in spontaneous contractions were seen with increasing concentrations of GYY4137, which also reduced underlying Ca transients. GYY4137 and NaHS significantly reduced oxytocin-stimulated and high-K depolarised contractions as well as spontaneous activity. Their inhibitory effects increased as gestation advanced, but were abruptly reversed in labour. Glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, abolished the inhibitory effect of GYY4137. These data suggest (i) H2S contributes to uterine quiescence from mid-gestation until labor, (ii) that H2S affects L-type calcium channels and KATP channels reducing Ca entry and thereby myometrial contractions, (iii) add to the evidence that H2S plays a physiological role in relaxing myometrium, and thus (iv) H2S is an attractive target for therapeutic manipulation of human myometrial contractility. PMID:23029460

Rapid adenosine release in the nucleus tractus solitarii during defence response in rats: real-time measurement in vivo

PubMed Central

Dale, Nicholas; Gourine, Alexander V; Llaudet, Enrique; Bulmer, David; Thomas, Teresa; Spyer, K Michael

2002-01-01

We have measured the release of adenosine and inosine from the dorsal surface of the brainstem and from within the nucleus tractus solitarii (NTS) during the defence response evoked by hypothalamic stimulation in the anaesthetised rat. At the surface of the brainstem, only release of inosine was detected on hypothalamic defence area stimulation. This inosine signal was greatly reduced by addition of the ecto-5′-nucleotidase inhibitor α,β-methylene ADP (200 μM), suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. By placing a microelectrode biosensor into the NTS under stereotaxic control we have recorded release of adenosine within this nucleus. By contrast to the brainstem surface, a fast increase in adenosine, accompanied only by a much smaller change in inosine levels, was seen following stimulation of the hypothalamic defence area. The release of adenosine following hypothalamic stimulation was mainly confined to a narrow region of the NTS some 500 μm in length around the level of the obex. Interestingly the release of adenosine was depletable: when the defence reaction was evoked at short time intervals, much less adenosine was released on the second stimulus. Our novel techniques have given unprecedented real-time measurement and localisation of adenosine release in vivo and demonstrate that adenosine is released at the right time and in sufficient quantities to contribute to the cardiovascular components of the defence reaction. PMID:12356888

TLR agonists stimulate Nlrp3-dependent IL-1β production independently of the purinergic P2X7 receptor in dendritic cells and in vivo.

PubMed

He, Yuan; Franchi, Luigi; Núñez, Gabriel

2013-01-01

On the basis of studies in mouse macrophages, activation of the nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain-containing 3 (Nlrp3) inflammasome is thought to require two signals. The first signal is provided by TLR stimulation and triggers the synthesis of the IL-1β precursor and Nlrp3. The second signal can be mediated by stimulation of the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) by millimolar concentrations of ATP. However, these high concentrations of ATP are not found normally in the in vivo extracellular milieu, raising concern about the physiological relevance of the ATP-P2X7 pathway of inflammasome activation. In this article, we show that unlike macrophages, murine bone marrow-derived and splenic dendritic cells (DCs) can secrete substantial amounts of mature IL-1β upon stimulation with TLR ligands in the absence of ATP stimulation. The differential ability of DCs to release IL-1β and activate caspase-1 was associated with increased expression of Nlrp3 under steady-state conditions and of pro-IL-1β and Nlrp3 after stimulation with TLR agonists. IL-1β secretion from stimulated DCs was largely dependent on the Nlrp3 inflammasome, but independent of P2X7 and unaffected by incubation with apyrase. More importantly, i.p. administration of LPS induced IL-1β production in serum, which was abrogated in Nlrp3-null mice but was unaffected in P2X7-deficient mice. These results demonstrate differential regulation of the Nlrp3 inflammasome in macrophages and DCs. Furthermore, they challenge the idea that the ATP-P2X7 axis is critical for TLR-induced IL-1β production via the Nlrp3 inflammasome in vivo.

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

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

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

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

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

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

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

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

21 CFR 864.7040 - Adenosine triphosphate release assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

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

Adenosine 5′-triphosphate (ATP) supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans

PubMed Central

2012-01-01

Background Nutritional supplements designed to increase adenosine 5′-triphosphate (ATP) concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity exercise. Oral ATP supplements have beneficial effects in some but not all studies examining physical performance. One of the remaining questions is whether orally administered ATP is bioavailable. We investigated whether acute supplementation with oral ATP administered as enteric-coated pellets led to increased concentrations of ATP or its metabolites in the circulation. Methods Eight healthy volunteers participated in a cross-over study. Participants were given in random order single doses of 5000 mg ATP or placebo. To prevent degradation of ATP in the acidic environment of the stomach, the supplement was administered via two types of pH-sensitive, enteric-coated pellets (targeted at release in the proximal or distal small intestine), or via a naso-duodenal tube. Blood ATP and metabolite concentrations were monitored by HPLC for 4.5 h (naso-duodenal tube) or 7 h (pellets) post-administration. Areas under the concentration vs. time curve were calculated and compared by paired-samples t-tests. Results ATP concentrations in blood did not increase after ATP supplementation via enteric-coated pellets or naso-duodenal tube. In contrast, concentrations of the final catabolic product of ATP, uric acid, were significantly increased compared to placebo by ~50% after administration via proximal-release pellets (P = 0.003) and naso-duodenal tube (P = 0.001), but not after administration via distal-release pellets. Conclusions A single dose of orally administered ATP is not bioavailable, and this may explain why several studies did not find ergogenic effects of oral ATP supplementation. On the other hand, increases in uric acid after release of ATP in the proximal

Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

PubMed

Schaffer, K; Herrmuth, H; Mueller, J; Coy, D H; Wong, H C; Walsh, J H; Classen, M; Schusdziarra, V; Schepp, W

1997-09-01

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2

Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

PubMed

Joseph, Elizabeth K; Green, Paul G; Bogen, Oliver; Alvarez, Pedro; Levine, Jon D

2013-02-13

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

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

DTIC Science & Technology

1986-01-01

Paddle and Burnstock (326), Williams and Forrester (463), Forrester and Williams (151) and Clemens and Forrester (82) provide evidence that hypoxia may...an ATp4 - receptor. Fed. Proc. 45:208, 1986. (abstr) 99. Dahlen , S.E. and Hedqvist, P. ATP, B,y-methylene ATP andN adenosine inhibit non-cholinergic...regulation of skeletal muscle blood low. Circ Res. 29:375-384, 1971. 117. Dodd, J., Jahr, C.E., Hamilton, P.N., Heath, M.J., Matthew , W.P., and Jessell, T.M

Experimental investigation of cavitation induced air release

NASA Astrophysics Data System (ADS)

Kowalski, Karoline; Pollak, Stefan; Hussong, Jeanette

Variations in cross-sectional areas may lead to pressure drops below a critical value, such that cavitation and air release are provoked in hydraulic systems. Due to a relatively slow dissolution of gas bubbles, the performance of hydraulic systems will be affected on long time scales by the gas phase. Therefore predictions of air production rates are desirable to describe the system characteristics. Existing investigations on generic geometries such as micro-orifice flows show an outgassing process due to hydrodynamic cavitation which takes place on time scales far shorter than diffusion processes. The aim of the present investigation is to find a correlation between global, hydrodynamic flow characteristics and cavitation induced undissolved gas fractions generated behind generic flow constrictions such as an orifice or venturi tube. Experimental investigations are realised in a cavitation channel that enables an independent adjustment of the pressure level upstream and downstream of the orifice. Released air fractions are determined by means of shadowgraphy imaging. First results indicate that an increased cavitation activity leads to a rapid increase in undissolved gas volume only in the choking regime. The frequency distribution of generated gas bubble size seems to depend only indirectly on the cavitation intensity driven by an increase of downstream coalescence events due to a more densely populated bubbly flow.

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

PubMed Central

Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

2014-01-01

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

Neuronal activity determines distinct gliotransmitter release from a single astrocyte

PubMed Central

Covelo, Ana

2018-01-01

Accumulating evidence indicates that astrocytes are actively involved in brain function by regulating synaptic activity and plasticity. Different gliotransmitters, such as glutamate, ATP, GABA or D-serine, released form astrocytes have been shown to induce different forms of synaptic regulation. However, whether a single astrocyte may release different gliotransmitters is unknown. Here we show that mouse hippocampal astrocytes activated by endogenous (neuron-released endocannabinoids or GABA) or exogenous (single astrocyte Ca2+ uncaging) stimuli modulate putative single CA3-CA1 hippocampal synapses. The astrocyte-mediated synaptic modulation was biphasic and consisted of an initial glutamate-mediated potentiation followed by a purinergic-mediated depression of neurotransmitter release. The temporal dynamic properties of this biphasic synaptic regulation depended on the firing frequency and duration of the neuronal activity that stimulated astrocytes. Present results indicate that single astrocytes can decode neuronal activity and, in response, release distinct gliotransmitters to differentially regulate neurotransmission at putative single synapses. PMID:29380725

A Role for the ATP7A Copper-transporting ATPase in Macrophage Bactericidal Activity*

PubMed Central

White, Carine; Lee, Jaekwon; Kambe, Taiho; Fritsche, Kevin; Petris, Michael J.

2009-01-01

Copper is an essential micronutrient that is necessary for healthy immune function. This requirement is underscored by an increased susceptibility to bacterial infection in copper-deficient animals; however, a molecular understanding of its importance in immune defense is unknown. In this study, we investigated the effect of proinflammatory agents on copper homeostasis in RAW264.7 macrophages. Interferon-γ was found to increase expression of the high affinity copper importer, CTR1, and stimulate copper uptake. This was accompanied by copper-stimulated trafficking of the ATP7A copper exporter from the Golgi to vesicles that partially overlapped with phagosomal compartments. Silencing of ATP7A expression attenuated bacterial killing, suggesting a role for ATP7A-dependent copper transport in the bactericidal activity of macrophages. Significantly, a copper-sensitive mutant of Escherichia coli lacking the CopA copper-transporting ATPase was hypersensitive to killing by RAW264.7 macrophages, and this phenotype was dependent on ATP7A expression. Collectively, these data suggest that copper-transporting ATPases, CopA and ATP7A, in both bacteria and macrophage are unique determinants of bacteria survival and identify an unexpected role for copper at the host-pathogen interface. PMID:19808669

The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells

PubMed Central

Castella, Barbara; Kopecka, Joanna; Sciancalepore, Patrizia; Mandili, Giorgia; Foglietta, Myriam; Mitro, Nico; Caruso, Donatella; Novelli, Francesco; Riganti, Chiara; Massaia, Massimo

2017-01-01

Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. PMID:28580927

Utility of a tripolar stimulating electrode for eliciting dopamine release in the rat striatum.

PubMed

Bergstrom, B P; Garris, P A

1999-03-01

The present study evaluated tripolar stimulating electrodes for eliciting dopamine release in the rat brain in vivo. Stimulating electrodes were placed either in the medial forebrain bundle or in the ventral mesencephalon associated with the ventral tegmental area and substantia nigra. The concentration of extracellular dopamine was monitored in dopamine terminal fields at 100-ms intervals using fast-scan cyclic voltammetry at carbon-fiber microelectrodes. To characterize the stimulated area, recordings were collected in several striatal regions including the caudate putamen and the core and shell of the nucleus accumbens. The tripolar electrode was equally effective in stimulating dopamine release in medial and lateral regions of the striatum. In contrast, responses evoked by a bipolar electrode were typically greater in one mediolateral edge versus the other. The added size of the tripolar electrode did not appear to cause complications as signals were stable over the course of the experiment (3 h). Subsets of mesostriatal dopamine neurons could also be selectively activated using the tripolar electrode in excellent agreement with previously described topography. Taken together, these results suggested that the tripolar stimulating electrode is well suited for studying the regulation of midbrain dopamine neurons in vivo.

Inhibition of presynaptic activity by zinc released from mossy fiber terminals during tetanic stimulation.

PubMed

Minami, Akira; Sakurada, Naomi; Fuke, Sayuri; Kikuchi, Kazuya; Nagano, Tetsuo; Oku, Naoto; Takeda, Atsushi

2006-01-01

Zinc exists in high densities in the giant boutons of hippocampal mossy fibers. On the basis of the evidence that zinc decreases extracellular glutamate concentration in the hippocampus, the presynaptic action of zinc released from mossy fibers during high-frequency (tetanic) stimulation was examined using hippocampal slices. The increase in zinc-specific fluorescent signals was observed in both extracellular and intracellular compartments in the mossy fiber terminals during the delivery of tetanic stimuli (100 Hz, 1 sec) to the dentate granule cell layer, suggesting that zinc released from mossy fibers is immediately retaken up by mossy fibers. When mossy fiber terminals were preferentially double-stained with zinc and calcium indicators and tetanic stimuli (100 Hz, 1 sec) were delivered to the dentate granule cell layer, the increase in calcium orange signal during the stimulation was enhanced in mossy fiber terminals by addition of CaEDTA, a membrane-impermeable zinc chelator, and was suppressed by addition of zinc. The decrease in FM4-64 signal (vesicular exocytosis) during tetanic stimulation (10 Hz, 180 sec), which induced mossy fiber long-term potentiation, was also enhanced in mossy fiber terminals by addition of CaEDTA and was suppressed by addition of zinc. The present study demonstrates that zinc released from mossy fibers may be a negative-feedback factor against presynaptic activity during tetanic stimulation.

Hypophosphatemia promotes lower rates of muscle ATP synthesis.

PubMed

Pesta, Dominik H; Tsirigotis, Dimitrios N; Befroy, Douglas E; Caballero, Daniel; Jurczak, Michael J; Rahimi, Yasmeen; Cline, Gary W; Dufour, Sylvie; Birkenfeld, Andreas L; Rothman, Douglas L; Carpenter, Thomas O; Insogna, Karl; Petersen, Kitt Falk; Bergwitz, Clemens; Shulman, Gerald I

2016-10-01

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [ 31 P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (V ATP ) and plasma inorganic phosphate (P i ) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent P i transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a -/- ) mice compared with their wild-type littermate controls. Rates of V ATP normalized in both hypophosphatemic groups after restoring plasma P i concentrations. Furthermore, V ATP was directly related to cellular and mitochondrial P i uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum P i content and muscle V ATP After oral P i repletion and normalization of serum P i levels, muscle V ATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood P i concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.-Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis. © The Author(s).

Energy transduction in the F1 motor of ATP synthase.

PubMed

Wang, H; Oster, G

1998-11-19

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central 'shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Energy transduction in the F1 motor of ATP synthase

NASA Astrophysics Data System (ADS)

Wang, Hongyun; Oster, George

1998-11-01

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism. This has been confirmed in the direction of hydrolysis, after isolation of the soluble F1 portion of the protein and visualization of the actual rotation of the central `shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F1 motor achieves its high mechanical torque and almost 100% efficiency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

PubMed

Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

2014-08-01

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

Stimulation by atropine of acetylcholine release and synthesis in cortical slices from rat brain

PubMed Central

Molenaar, P. C.; Polak, R. L.

1970-01-01

1. Cortical slices from rat brain were incubated in media containing the irreversible cholinesterase inhibitor soman and a high KCl concentration, and the release and synthesis of acetylcholine (ACh) were determined. 2. Atropine enhanced the release and synthesis of ACh. 3. Tetrodotoxin, a substance which blocks nervous conduction, did not influence the release and synthesis of ACh, in the absence or in the presence of atropine. Therefore the nerve endings are probably the site at which atropine acts when stimulating the release and synthesis of ACh. 4. Pretreatment of the slices with botulinum type A toxin partially blocked the release and synthesis of ACh and reduced the extra amounts of ACh released and synthesized under the influence of atropine. 5. Lowering the calcium or raising the magnesium concentration in the incubation medium reduced the release and synthesis of ACh and their enhancement by atropine. 6. Physostigmine decreased the total extractable ACh content of the slices during incubation in a 25 mM KCl containing medium. This decrease was nearly prevented when the release and synthesis of ACh were inhibited by omission of the calcium ions from the medium, but was enhanced by atropine. 7. The observations made with pretreatment by botulinum type A toxin, with changes in the calcium and magnesium concentration as well as with physostigmine, all support the theory that it is primarily the release of ACh which is enhanced by atropine and that its stimulating action on the synthesis results from the increased release. PMID:5497792

Enhanced hydrogen peroxide release from macrophages stimulated with streptococcal preparation OK-432.

PubMed Central

Saito, H; Tomioka, H

1979-01-01

Wheat germ lectin was found to be a potent triggering agent for hydrogen peroxide release from mouse peritoneal macrophages. Macrophages stimulated by intraperitoneal injection of OK-432, a lyophilized attenuated streptococcal preparation, were highly responsive to wheat germ lectin. PMID:546795

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.

Single K ATP channel opening in response to action potential firing in mouse dentate granule neurons.

PubMed

Tanner, Geoffrey R; Lutas, Andrew; Martínez-François, Juan Ramón; Yellen, Gary

2011-06-08

ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP, Caffeine, Quinine, and Local Anesthetics

PubMed Central

Carvalho, Arselio P.

1968-01-01

Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10-5 – 10-4 M ATP to exchange with 45Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on 45Ca fluxes and on the twitch output observed in whole muscles. PMID:19873636

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

PubMed Central

Sukhareva, M; Morrissette, J; Coronado, R

1994-01-01

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine

Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

PubMed Central

Lembong, Josephine; Sabass, Benedikt; Sun, Bo; Rogers, Matthew E.; Stone, Howard A.

2015-01-01

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young's modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca2+ oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell–cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment. PMID:26063818

Oleic acid stimulates glucagon-like peptide-1 release from enteroendocrine cells by modulating cell respiration and glycolysis.

PubMed

Clara, Rosmarie; Langhans, Wolfgang; Mansouri, Abdelhak

2016-03-01

Glucagon-like peptide-1 (GLP-1) is a potent satiating and incretin hormone released by enteroendocrine L-cells in response to eating. Dietary fat, in particular monounsaturated fatty acids, such as oleic acid (OA), potently stimulates GLP-1 secretion from L-cells. It is, however, unclear whether the intracellular metabolic handling of OA is involved in this effect. First we determined the optimal medium for the bioenergetics measurements. Then we examined the effect of OA on the metabolism of the immortalized enteroendocrine GLUTag cell model and assessed GLP-1 release in parallel. We measured oxygen consumption rate and extracellular acidification rate in response to OA and to different metabolic inhibitors with the Seahorse extracellular flux analyzer. OA increased cellular respiration and potently stimulated GLP-1 release. The fatty acid oxidation inhibitor etomoxir did neither reduce OA-induced respiration nor affect the OA-induced GLP-1 release. In contrast, inhibition of the respiratory chain or of downstream steps of aerobic glycolysis reduced the OA-induced GLP-1 release, and an inhibition of the first step of glycolysis by addition of 2-deoxy-d-glucose even abolished it. These findings indicate that an indirect stimulation of glycolysis is crucial for the OA-induced release of GLP-1. Copyright © 2015 Elsevier Inc. All rights reserved.

Modeling steady state SO2-dependent changes in capillary ATP concentration using novel O2 micro-delivery methods

PubMed Central

Ghonaim, Nour W.; Fraser, Graham M.; Ellis, Christopher G.; Yang, Jun; Goldman, Daniel

2013-01-01

Adenosine triphosphate (ATP) is known to be released from the erythrocyte in an oxygen (O2) dependent manner. Since ATP is a potent vasodilator, it is proposed to be a key regulator in the pathway that mediates micro-vascular response to varying tissue O2 demand. We propose that ATP signaling mainly originates in the capillaries due to the relatively long erythrocyte transit times in the capillary and the short ATP diffusion distance to the electrically coupled endothelium. We have developed a computational model to investigate the effect of delivering or removing O2 to limited areas at the surface of a tissue with an idealized parallel capillary array on total ATP concentration. Simulations were conducted when exposing full surface to perturbations in tissue O2 tension (PO2) or locally using a circular micro-outlet (~100 μm in diameter), a square micro-slit (200 × 200 μm), or a rectangular micro-slit (1000 μm wide × 200 μm long). Results indicated the rectangular micro-slit has the optimal dimensions for altering hemoglobin saturations (SO2) in sufficient number capillaries to generate effective changes in total [ATP]. This suggests a threshold for the minimum number of capillaries that need to be stimulated in vivo by imposed tissue hypoxia to induce a conducted micro-vascular response. SO2 and corresponding [ATP] changes were also modeled in a terminal arteriole (9 μm in diameter) that replaces 4 surface capillaries in the idealized network geometry. Based on the results, the contribution of terminal arterioles to the net change in [ATP] in the micro-vascular network is minimal although they would participate as O2 sources thus influencing the O2 distribution. The modeling data presented here provide important insights into designing a novel micro-delivery device for studying micro-vascular O2 regulation in the capillaries in vivo. PMID:24069001

ATP-sensitive potassium channels participate in glucose uptake in skeletal muscle and adipose tissue.

PubMed

Miki, Takashi; Minami, Kohtaro; Zhang, Li; Morita, Mizuo; Gonoi, Tohru; Shiuchi, Tetsuya; Minokoshi, Yasuhiko; Renaud, Jean-Marc; Seino, Susumu

2002-12-01

ATP-sensitive potassium (K(ATP)) channels are known to be critical in the control of both insulin and glucagon secretion, the major hormones in the maintenance of glucose homeostasis. The involvement of K(ATP) channels in glucose uptake in the target tissues of insulin, however, is not known. We show here that Kir6.2(-/-) mice lacking Kir6.2, the pore-forming subunit of these channels, have no K(ATP) channel activity in their skeletal muscles. A 2-deoxy-[(3)H]glucose uptake experiment in vivo showed that the basal and insulin-stimulated glucose uptake in skeletal muscles and adipose tissues of Kir6.2(-/-) mice is enhanced compared with that in wild-type (WT) mice. In addition, in vitro measurement of glucose uptake indicates that disruption of the channel increases the basal glucose uptake in Kir6.2(-/-) extensor digitorum longus and the insulin-stimulated glucose uptake in Kir6.2(-/-) soleus muscle. In contrast, glucose uptake in adipose tissue, measured in vitro, was similar in Kir6.2(-/-) and WT mice, suggesting that the increase in glucose uptake in Kir6.2(-/-) adipocytes is mediated by altered extracellular hormonal or neuronal signals altered by disruption of the K(ATP) channels.

Thyroid hormone stimulates progesterone release from human luteal cells by generating a proteinaceous factor.

PubMed

Datta, M; Roy, P; Banerjee, J; Bhattacharya, S

1998-09-01

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.

PREVENTION REFERENCE MANUAL: CONTROL TECHNOLOGIES, VOL. 2. POST-RELEASE MITIGATION MEASURES FOR CONTROLLING ACCIDENTAL RELEASES OF AIR TOXICS

EPA Science Inventory

The volume discusses prevention and protection measures for controlling accidental releases of air toxics. The probability of accidental releases depends on the extent to which deviations (in magnitude and duration) in the process can be tolerated before a loss of chemical contai...

Rosiglitazone stimulates the release and synthesis of insulin by enhancing GLUT-2, glucokinase and BETA2/NeuroD expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyo-Sup; Noh, Jung-Hyun; Hong, Seung-Hyun

2008-03-14

Peroxisome proliferator-activated receptor (PPAR)-{gamma} is a member of the nuclear receptor superfamily, and its ligands, the thiazolidinediones, might directly stimulate insulin release and insulin synthesis in pancreatic {beta}-cells. In the present study, we examined the effects of rosiglitazone (RGZ) on insulin release and synthesis in pancreatic {beta}-cell (INS-1). Insulin release and synthesis were stimulated by treatment with RGZ for 24 h. RGZ upregulated the expressions of GLUT-2 and glucokinase (GCK). Moreover, it was found that RGZ increased the expression of BETA2/NeuroD gene which could regulate insulin gene expression. These results suggest that RGZ could stimulate the release and synthesis ofmore » insulin through the upregulation of GLUT-2, GCK, and BETA2/NeuroD gene expression.« less

Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.

PubMed

Stenqvist, Johanna; Winder, Michael; Carlsson, Thomas; Aronsson, Patrik; Tobin, Gunnar

2017-08-15

Both acetylcholine and adenosine 5'-triphosphate (ATP) are released from the urothelium. In in vivo experiments ATP has been shown to evoke contractile responses that are significantly reduced by atropine. Currently, we aimed to examine the cholinergic part of the ATP-evoked contractile response of normal and inflamed (cyclophosphamide-treated rats) bladders. A whole bladder preparation that enabled drug administration either outside or inside the urinary bladder was used. The responses were examined in bladders from control and cyclophosphamide-treated rats that were either intact or urothelium-denuded. The expression of choline acetyltransferase and carnitine acetyltransferase were examined by Western blotting of normal and inflamed bladders. Methacholine evoked larger contractions when administered to the outside of the bladder in comparison to instillation. For ATP, an opposite trend emerged. While atropine substantially reduced the ATP-induced responses at internal administration (7.4±1.1 and 3.7±0.9 mN at 10 -3 M; n=13; P<0.001), it had no effect when administered outside the bladder. The removal of the urothelium caused a similar reduction of the responses to internal administration of ATP as caused by atropine. In cyclophosphamide-treated rats, neither atropine nor urothelium-denudation had any effect on the ATP-evoked responses. No changes in the expressions of the acetylcholine synthesising enzymes were observed. The current study shows that ATP induces a release of urothelial acetylcholine that contributes to the purinergic contractile response in the rat urinary bladder. This atropine-sensitive part of the purinergic contractile response is absent in the inflamed bladder. This may be one pathological mechanism involved in bladder dysfunction. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased glutamate-stimulated norepinephrine release from prefrontal cortex slices of spontaneously hypertensive rats.

PubMed

Russell, V A; Wiggins, T M

2000-12-01

Spontaneously hypertensive rats (SHR) have behavioral characteristics (hyperactivity, impulsiveness, poorly sustained attention) similar to the behavioral disturbances of children with attention-deficit hyperactivity disorder (ADHD). We have previously shown that dopaminergic and noradrenergic systems are disturbed in the prefrontal cortex of SHR compared to their normotensive Wistar-Kyoto (WKY) control rats. It was of interest to determine whether the underlying neural circuits that use glutamate as a neurotransmitter function normally in the prefrontal cortex of SHR. An in vitro superfusion technique was used to demonstrate that glutamate caused a concentration-dependent stimulation of [3H]norepinephrine release from rat prefrontal cortex slices. Glutamate (100 microM and 1 mM) caused significantly greater release of norepinephrine from prefrontal cortex slices of SHR than from control slices. The effect of glutamate was not mediated by NMDA receptors, since NMDA (10 and 100 microM) did not exert any effect on norepinephrine release and MK-801 (10 microM) did not antagonize the effect of 100 microM glutamate. These results demonstrate that glutamate stimulates norepinephrine release from rat prefrontal cortex slices and that this increase is enhanced in SHR. The results are consistent with the suggestion that the noradrenergic system is overactive in prefrontal cortex of SHR, the animal model for ADHD.

Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

PubMed

Tripathi, Diwaker; Zhang, Tong; Koo, Abraham J; Stacey, Gary; Tanaka, Kiwamu

2018-01-01

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis ( Arabidopsis thaliana ) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1 - 3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1 , but not in the JA biosynthesis mutant, aos , or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses. © 2018

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

PubMed Central

Schneider, H C; Westermann, B; Neupert, W; Brunner, M

1996-01-01

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle. Images PMID:8918457

Adrenocorticotropic Hormone Suppresses Gonadotropin-Stimulated Estradiol Release from Zebrafish Ovarian Follicles

PubMed Central

Alsop, Derek; Ings, Jennifer S.; Vijayan, Mathilakath M.

2009-01-01

While stress is known to impact reproductive performance, the pathways involved are not entirely understood. Corticosteroid effects on the functioning of the hypothalamus-pituitary-gonadal axis are thought to be a key aspect of stress-mediated reproductive dysfunction. A vital component of the stress response is the pituitary secretion of adrenocorticotropic hormone (ACTH), which binds to the melanocortin 2 receptor (MC2R) in the adrenal glands and activates cortisol biosynthesis. We recently reported MC2R mRNA abundance in fish gonads leading to the hypothesis that ACTH may be directly involved in gonadal steroid modulation. Using zebrafish (Danio rerio) ovarian follicles, we tested the hypothesis that acute ACTH stimulation modulates cortisol and estradiol (E2) secretion. ACTH neither affected cortisol nor unstimulated E2 release from ovarian follicles. However, ACTH suppressed human chorionic gonadotropin (hCG)-stimulated E2 secretion in a dose-related manner, with a maximum decrease of 62% observed at 1 I.U. ACTH mL−1. This effect of ACTH on E2 release was not observed in the presence of either 8-bromo-cAMP or forskolin, suggesting that the mechanism(s) involved in steroid attenuation was upstream of adenylyl cyclase activation. Overall, our results suggest that a stress-induced rise in plasma ACTH levels may initiate a rapid down-regulation of acute stimulated E2 biosynthesis in the zebrafish ovary, underscoring a novel physiological role for this pituitary peptide in modulating reproductive activity. PMID:19649243

Cytidine 5'-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes.

PubMed Central

Tyrsted, G; Gamulin, V

1979-01-01

The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h. PMID:424294

Activation of p38 in C2C12 myotubes following ATP depletion depends on extracellular glucose.

PubMed

Hsu, Chia George; Burkholder, Thomas J

2015-06-01

Muscle cells adjust their glucose metabolism in response to myriad stimuli, and particular attention has been paid to glucose metabolism after contraction, ATP depletion, and insulin stimulation. Each of these requires translocation of GLUT4 to the cell membrane, and may require activation of glucose transporters by p38. In contrast, AICAR stimulates glucose transport without activation of p38, suggesting that p38 activation may be an indirect consequence of accelerated glucose transport or metabolism. This study was designed to investigate the contribution of AMPK and p38 to ATP homeostasis and glucose metabolism to test the hypothesis that p38 reflects glycolytic activity rather than controls glucose uptake. Treating mature myotubes with rotenone caused transient ATP depletion in 15 min with recovery by 120 min, associated with increased lactate production. Both ACC and p38 were rapidly phosphorylated, but ACC remained phosphorylated while p38 phosphorylation declined as ATP recovered. AMPK inhibition blocked ATP recovery, lactate production, and phosphorylation of p38 and ACC. Inhibition of p38 had little effect. AICAR induced ACC phosphorylation, but not lactate production or p38 phosphorylation. Finally, removing extracellular glucose potentiated rotenone-induced AMPK activation, but reduced lactate generation, ATP recovery and p38 activation. Thus, glucose metabolism is highly sensitive to ATP homeostasis via AMPK activity, but p38 activity is dispensable. Although p38 is strongly phosphorylated during ATP depletion, this appears to be an indirect consequence of accelerated glycolysis.

Ultrasound stimulated release of gallic acid from chitin hydrogel matrix.

PubMed

Jiang, Huixin; Kobayashi, Takaomi

2017-06-01

Ultrasound (US) stimulated drug release was examined in this study using a chitin hydrogel matrix loaded with gallic acid (GA), a drug used for wound healing and anticancer. Using phase inversion, GA-chitin hydrogels were prepared from chitin-dimethylacetamide (DMAc)/lithium chloride (LiCl) solution in the presence of GA, with 24h exposure of the solution to water vapor. The GA release from the GA-chitin hydrogel was examined under different US powers of 0-30W at 43kHz. The effects of GA loading amounts in the hydrogels (0.54, 0.43, and 0.25mg/cm 3 ) and chitin concentrations (0.1, 0.5, and 1wt%) on the release behaviors were recorded under 43kHz US exposure at 30W. Results show that US accelerated the release efficiencies for all samples. Furthermore, the release efficiency increased concomitantly with increasing US power, GA loading amount, and decrease of the chitin concentration. The highest release rate of 0.74μg/mL·min was obtained from 0.54mg/cm 3 of GA-loaded hydrogel fabricated from a 0.1wt% chitin mixture solution under 43kHz US exposure at 30W: nine times higher than that of the sample without US exposure. The hydrogel viscoelasticity demonstrated that the US irradiation rigidified the material. FT-IR showed that US can break the hydrogen bonds in the GA-chitin hydrogels. Copyright © 2017 Elsevier B.V. All rights reserved.

Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling

PubMed Central

Moehring, Francie; Cowie, Ashley M; Menzel, Anthony D; Weyer, Andy D; Grzybowski, Michael; Arzua, Thiago; Geurts, Aron M; Palygin, Oleg

2018-01-01

The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions. PMID:29336303

Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schubert,H.; Hill, C.

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, therebymore » 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.« less

P2 receptor stimulation induces amyloid precursor protein production and secretion in rat cortical astrocytes.

PubMed

Tran, Minh D

2011-04-04

Amyloid precursor protein (APP) is ubiquitously expressed in a variety of tissues but is predominantly expressed in the brain. The expression of APP has been well studied in neurons but little is known about its presence in astrocytes. The study presented here shows that purinergic signaling is involved in the production and secretion of APP in primary cultures of rat cortical astrocytes. Extracellular ATP caused an increase in APP production and release in a time- and concentration-dependent manner and was inhibited by antagonists of P2 receptors. Further agonist and antagonist studies revealed involvement of P2Y2 and P2Y4 receptors in nucleotide-stimulated production and release of APP. In addition, signaling studies with various protein kinase inhibitors demonstrated that blockade of mitogen-activated protein kinases, but not Akt, inhibited nucleotide-stimulated APP expression and release. These results indicate that APP production and secretion can be regulated by activation of P2Y2/4 receptors coupled to protein kinase signaling pathways and suggest that astrocytes can be a potential source of APP. Published by Elsevier Ireland Ltd.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

PubMed

Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

2016-02-01

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. Published by Elsevier Inc.

ATP-Responsive and Near-Infrared-Emissive Nanocarriers for Anticancer Drug Delivery and Real-Time Imaging.

PubMed

Qian, Chenggen; Chen, Yulei; Zhu, Sha; Yu, Jicheng; Zhang, Lei; Feng, Peijian; Tang, Xin; Hu, Quanyin; Sun, Wujin; Lu, Yue; Xiao, Xuanzhong; Shen, Qun-Dong; Gu, Zhen

2016-01-01

Stimuli-responsive and imaging-guided drug delivery systems hold vast promise for enhancement of therapeutic efficacy. Here we report an adenosine-5'-triphosphate (ATP)-responsive and near-infrared (NIR)-emissive conjugated polymer-based nanocarrier for the controlled release of anticancer drugs and real-time imaging. We demonstrate that the conjugated polymeric nanocarriers functionalized with phenylboronic acid tags on surface as binding sites for ATP could be converted to the water-soluble conjugated polyelectrolytes in an ATP-rich environment, which promotes the disassembly of the drug carrier and subsequent release of the cargo. In vivo studies validate that this formulation exhibits promising capability for inhibition of tumor growth. We also evaluate the metabolism process by monitoring the fluorescence signal of the conjugated polymer through the in vivo NIR imaging.

A lipid switch unlocks Parkinson’s disease-associated ATP13A2

PubMed Central

Holemans, Tine; Sørensen, Danny Mollerup; van Veen, Sarah; Martin, Shaun; Hermans, Diane; Kemmer, Gerdi Christine; Van den Haute, Chris; Baekelandt, Veerle; Günther Pomorski, Thomas; Agostinis, Patrizia; Wuytack, Frank; Palmgren, Michael; Eggermont, Jan; Vangheluwe, Peter

2015-01-01

ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders. PMID:26134396

Intravascular ATP and the regulation of blood flow and oxygen delivery in humans.

PubMed

Crecelius, Anne R; Kirby, Brett S; Dinenno, Frank A

2015-01-01

Regulation of vascular tone is a complex response that integrates multiple signals that allow for blood flow and oxygen supply to match oxygen demand appropriately. Here, we discuss the potential role of intravascular adenosine triphosphate (ATP) as a primary factor in these responses and put forth the hypothesis that deficient ATP release contributes to impairments in vascular control exhibited in aged and diseased populations.

Phorbol ester stimulates calcium sequestration in saponized human platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, K.; Nachmias, V.T.

1987-11-25

When platelets are activated by agonists, calcium (Ca2+) is released from an intracellular storage site. Recent studies using fura-2 show that, after thrombin stimulation, the rise in free calcium is transient and returns to base-line levels in 2-3 min, while the transient following ADP stimulation lasts only 15-20 s. We reported previously that the phorbol ester 12,13-phorbol myristate acetate (PMA), added at nanomolar levels after thrombin, immediately accelerated the rate of return of calcium to the base line severalfold. In the present study, we used both intact and saponized platelets to determine whether this is due to stimulation of calciummore » sequestration. Using fura-2 and intact platelets, we found 1) that PMA stimulated the restoration of free Ca2+ levels after ADP as well as after thrombin, and 2) that H-7, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), slowed the return of Ca2+ to baseline levels. Using saponized platelets, we also found 3) that pretreatment of platelets with PMA before saponin treatment increased the ATP-dependent /sup 45/Ca2+ uptake 2-fold, with a half-maximal effect at 5 nm; 4) that most of the Ca2+ released by ionomycin or by myoinositol 1,4,5-trisphosphate; and 5) that a GTP-binding protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), decreased basal or PMA-stimulated /sup 45/Ca2+ uptake in saponin-treated platelets. Our data suggest that activation of protein kinase C stimulates the sequestration of Ca2+ independently of cAMP or myoinositol 1,4,5-trisphosphate.« less

Optimal positions for the release of primitive neonatal reflexes stimulating breastfeeding.

PubMed

Colson, Suzanne D; Meek, Judith H; Hawdon, Jane M

2008-07-01

Despite widespread skills-teaching, 37% of UK mothers initiating breastfeeding stop by six weeks suggesting a need to reappraise current support strategies. Rooting, sucking and swallowing have been studied extensively but little is known about the role other primitive neonatal reflexes (PNRs) might play to support breastfeeding. To describe and compare PNRs observed during feeding, investigating whether certain feeding behaviours and positions, collectively termed Biological Nurturing, (BN) are associated with the release of those reflexes pivotal in establishing successful feeding. 40 breastfed healthy term mother/baby pairs were recruited using quota sampling to stratify term gestational age. Feeding sessions were videotaped in the first postnatal month, either in hospital or at home. 20 PNRs were validated and classified into 4 types (endogenous, motor, rhythmic and anti-gravity) and 2 functional clusters (finding/latching, milk transfer) either stimulating or hindering feeding. Significantly more PNRs were observed as stimulants in semi-reclined postures (BN) than when mothers were upright or side-lying (p=<0.0005). This study is the first to describe a range of semi-reclined maternal postures interacting with neonatal positions, releasing maternal instinctual behaviours and PNRs stimulating breastfeeding. Traditionally the human neonate has been considered a dorsal feeder with pressure needed along the baby's back. Compelling visual data here illustrate that the newborn is an abdominal feeder and, like some other animals, displays anti-gravity reflexes aiding latch. Findings suggest that breastfeeding initiation is innate for both mother and baby, not learned, thus challenging the routine skills-teaching currently central to breastfeeding support.

Improved fault tolerance for air bag release in automobiles

NASA Astrophysics Data System (ADS)

Yeshwanth Kumar, C. H.; Prudhvi Prasad, P.; Uday Shankar, M.; Shanmugasundaram, M.

2017-11-01

In order to increase the reliability of the airbag system in automobiles which in turn increase the safety of the automobile we require improved airbag release system, our project deals with Triple Modular Redundancy (TMR) Technique where we use either three Sensors interfaced with three Microcontrollers which given as input to the software voter which produces majority output which is feed to the air compressor for releasing airbag. This concept was being used, in this project we are increasing reliability and safety of the entire system.

P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity.

PubMed

Tanigawa, Hitoshi; Toyoda, Futoshi; Kumagai, Kosuke; Okumura, Noriaki; Maeda, Tsutomu; Matsuura, Hiroshi; Imai, Shinji

2018-05-29

Extracellular ATP regulates various cellular functions by engaging multiple subtypes of P2 purinergic receptors. In many cell types, the ionotropic P2X7 receptor mediates pathological events such as inflammation and cell death. However, the importance of this receptor in chondrocytes remains largely unexplored. Here, we report the functional identification of P2X7 receptor in articular chondrocytes and investigate the involvement of P2X7 receptors in ATP-induced cytotoxicity. Chondrocytes were isolated from rabbit articular cartilage, and P2X7 receptor currents were examined using the whole-cell patch-clamp technique. ATP-induced cytotoxicity was evaluated by measuring caspase-3/7 activity, lactate dehydrogenase (LDH) leakage, and prostagrandin E 2 (PGE 2 ) release using microscopic and fluorimetric/colorimetric evaluation. Extracellular ATP readily evoked a cationic current without obvious desensitization. This ATP-activated current was dose related, but required millimolar concentrations. A more potent P2X7 receptor agonist, BzATP, also activated this current but at 100-fold lower concentrations. ATP-induced currents were largely abolished by selective P2X7 antagonists, suggesting a predominant role for the P2X7 receptor. RT-PCR confirmed the presence of P2X7 in chondrocytes. Heterologous expression of a rabbit P2X7 clone successfully reproduced the ATP-induced current. Exposure of chondrocytes to ATP increased caspase-3/7 activities, an effect that was totally abrogated by P2X7 receptor antagonists. Extracellular ATP also enhanced LDH release, which was partially attenuated by the P2X7 inhibitor. The P2X7 receptor-mediated elevation in apoptotic caspase signaling was accompanied by increased PGE 2 release and was attenuated by inhibition of either phospholipase A 2 or cyclooxygenase-2. This study provides direct evidence for the presence of functional P2X7 receptors in articular chondrocytes. Our results suggest that the P2X7 receptor is a potential therapeutic

Protecting muscle ATP: positive roles for peripheral defense mechanisms-introduction.

PubMed

Myburgh, Kathryn H

2004-01-01

Skeletal muscle has evolved an impressive array of mechanisms for peripherally mediated control of ATP homeostasis. Some of these mechanisms are intracellular, and others are extracellular and include influences on the cross-bridge cycle itself and substrate supply. This paper introduces three distinctly different topics that nevertheless all have ATP defense in common. The role of ADP in fatigue is controversial but has recently been more clearly delineated so that an effect on alleviating force declines during extreme fatigue is plausible. AMP plays its role by activating the protein-kinase, AMPK, which is a key sensor of cellular energy stress. AMPK has different isoforms, is not uniformly distributed in the cell, and its activation is carefully controlled. It has multiple effects including improvements in substrate supply for the metabolic pathways producing ATP and inhibition of anabolic processes to further spare ATP. Red blood cells have the capacity to sense hypoxia and to release vasodilators where there is a locally increased demand for blood supply. The papers in this series emphasize the important positive roles of metabolites and sensors of fatigue in the balance between ATP supply and demand.

Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cronstein, B.N.; Eberle, M.A.; Levin, R.I.

1991-03-15

Although commonly used to control a variety of inflammatory diseases, the mechanism of action of a low dose of methotrexate remains a mystery. Methotrexate accumulates intracellularly where it may interfere with purine metabolism. Therefore, the authors determined whether a 48-hr pretreatment with methotrexate affected adenosine release from ({sup 14}C)adenine-labeled human fibroblasts and umbilical vein endothelial cells. Methotrexate significantly increased adenosine release by fibroblasts. The effect of methotrexate on adenosine release was not due to cytotoxicity since cells treated with maximal concentrations of methotrexate took up ({sup 14}C)adenine and released {sup 14}C-labeled purine (a measure of cell injury) in a mannermore » identical to control cells. Methotrexate treatment of fibroblasts dramatically inhibited adherence to fibroblasts by both unstimulated neutrophils and stimulated neutrophils. One hypothesis that explains the effect of methotrexate on adenosine release is that, by inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, methotrexate induces the accumulation of AICAR, the nucleoside precursor of which has previously been shown to cause adenosine release from ischemic cardiac tissue. The observation that the antiinflammatory actions of methotrexate are due to the capacity of methotrexate to induce adenosine release may form the basis for the development of an additional class of antiinflammatory drugs.« less

Rupatadine inhibits inflammatory mediator release from human laboratory of allergic diseases 2 cultured mast cells stimulated by platelet-activating factor.

PubMed

Alevizos, Michail; Karagkouni, Anna; Vasiadi, Magdalini; Sismanopoulos, Nikolaos; Makris, Michael; Kalogeromitros, Dimitrios; Theoharides, Theoharis C

2013-12-01

Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 μmol/L) and substance P (1 μmol/L) with or without pretreatment with RUP (2.5 and 25 μmol/L), which was added 10 minutes before stimulation. Release of β-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001-0.1 μmol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 μmol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 μmol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation. Copyright © 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The effect of medium viscosity on kinetics of ATP hydrolysis by the chloroplast coupling factor CF1.

PubMed

Malyan, Alexander N

2016-05-01

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

Thrombin stimulates increased plasminogen activator inhibitor-1 release from liver compared to lung endothelium.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Gonzalez, Eduardo; Kelher, Marguerite R; Sauaia, Angela; Banerjee, Anirban; Silliman, Christopher C

2018-05-01

Plasminogen activator inhibitor-1 (PAI-1) is a major regulator of the fibrinolytic system, covalently binding to tissue plasminogen activator and blocking its activity. Fibrinolysis shutdown is evident in the majority of severely injured patients in the first 24 h and is thought to be due to PAI-1. The source of this PAI-1 is thought to be predominantly endothelial cells, but there are known organ-specific differences, with higher levels thought to be in the liver. Thrombin generation is also elevated in injured patients and is a potent stimulus for PAI-1 release in human umbilical endothelial cells. We hypothesize that thrombin induces liver endothelial cells to release increased amounts of PAI-1, versus pulmonary endothelium, consisting of both stored PAI-1 and a larger contribution from de novo PAI-1 synthesis. Human liver sinusoidal endothelial cells (LSECs) and human microvascular lung endothelial cells (HMVECs) were stimulated in vitro ± thrombin (1 and 5 IU/mL) for 15-240 min, the supernatants were collected, and PAI-1 was measured by enzyme-linked immunosorbent assays. To elucidate the PAI-1 contribution from storage versus de novo synthesis, cycloheximide (10 μg/mL) was added before thrombin in separate experiments. While both LSECs and HMVECs rapidly stimulated PAI-1 release, LSECs released more PAI-1 than HMVECs in response to high-dose thrombin, whereas low-dose thrombin did not provoke immediate release. LSECs continued to release PAI-1 over the ensuing 240 min, whereas HMVECs did not. Cycloheximide did not inhibit early PAI-1 release from LSECs but did at the later time points (30-240 min). Thrombin elicits increased amounts of PAI-1 release from liver endothelium compared with lung, with a small presynthesized stored contribution and a later, larger increase in PAI-1 release via de novo synthesis. This study suggests that the liver may be an important therapeutic target for inhibition of the hypercoagulable surgical patient and the

Human hair follicles contain two forms of ATP-sensitive potassium channels, only one of which is sensitive to minoxidil.

PubMed

Shorter, Katie; Farjo, Nilofer P; Picksley, Steven M; Randall, Valerie A

2008-06-01

Hair disorders cause psychological distress but are generally poorly controlled; more effective treatments are required. Despite the long-standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP-sensitive potassium (K(ATP)) channels. To investigate whether K(ATP) channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tolbutamide, a K(ATP) channel blocker, opposed each other's effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase-polymerase chain reaction identified K(ATP) channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore-forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were examined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhibited SUR2B and Kir6.1. Immunohistochemistry demonstrated similar protein distributions. Thus, human follicles respond biologically to K(ATP) channel regulators in culture and express genes and proteins for two K(ATP) channels, Kir6.2/SUR1 and Kir6.1/SUR2B; minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain K(ATP) channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.

Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ɛ Subunit

PubMed Central

Keis, Stefanie; Stocker, Achim; Dimroth, Peter; Cook, Gregory M.

2006-01-01

The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ɛ subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F1 mutant with either a truncated ɛ subunit [i.e., TA2F1(ɛΔC)] or substitution of basic residues in the second α-helix of ɛ with nonpolar alanines [i.e., TA2F1(ɛ6A)] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F1Fo-ATP synthase and TA2F1(ɛWT) complex with proteases revealed that the ɛ subunit was resistant to proteolytic digestion. In contrast, the ɛ subunit of TA2F1(ɛ6A) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ɛ was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ɛ subunit in the entire holoenzyme, we reconstituted recombinant TA2F1 complexes with F1-stripped native membranes of strain TA2.A1. The reconstituted TA2FoF1(ɛWT) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F1Fo-ATP synthase in native membranes. Reconstituted TA2FoF1(ɛ6A) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the �

Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma.

PubMed Central

Lyle, S; Geller, D H; Ng, K; Westley, J; Schwartz, N B

1994-01-01

ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction. PMID:8042976

Unexpected Role of the Copper Transporter ATP7A in PDGF-Induced Vascular Smooth Muscle Cell Migration

PubMed Central

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D.; Maryon, Edward B.; Kaplan, Jack H.; Ushio-Fukai, Masuko; Fukai, Tohru

2010-01-01

Rationale Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1, but also by the copper exporter ATP7A (Menke ATPase) whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. Objective To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Methods and Results Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A siRNA or CTR siRNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor pro-lysyl oxidase (Pro-LOX) in lipid raft fraction as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based X-ray fluorescence microscopy at neointimal VSMCs in wire injury model. Conclusions These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis. PMID:20671235

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

PubMed

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D; Maryon, Edward B; Kaplan, Jack H; Ushio-Fukai, Masuko; Fukai, Tohru

2010-09-17

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells*

PubMed Central

Dai, Feihan F.; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B.

2015-01-01

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H+-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. PMID:26272612

Kisspeptin stimulates growth hormone release by utilizing Neuropeptide Y pathways and is dependent on the presence of ghrelin

USDA-ARS?s Scientific Manuscript database

Although kisspeptin is the primary stimulator of gonadotropin releasing hormone secretion and therefore the hypothalamic-pituitary gonadal axis, new findings suggest kisspeptin can also regulate additional neuroendocrine processes including release of growth hormone (GH). Central delivery of kisspep...

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.

Modulation of extracellular ATP content of mast cells and DRG neurons by irradiation: studies on underlying mechanism of low-level-laser therapy.

PubMed

Wang, Lina; Hu, Lei; Grygorczyk, Ryszard; Shen, Xueyong; Schwarz, Wolfgang

2015-01-01

Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

PubMed Central

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

Purinergic and cholinergic neuro-neuronal transmission underlying reflexes activated by mucosal stimulation in the isolated guinea-pig ileum

PubMed Central

Spencer, Nick J; Walsh, Michelle; Smith, Terence K

2000-01-01

stimulation releases ACh and ATP in both ascending and descending excitatory reflex pathways that synapse with excitatory motoneurons to the LM and CM. PMID:10639107

Bimodal Action of Protons on ATP Currents of Rat PC12 Cells

PubMed Central

Skorinkin, Andrei; Nistri, Andrea; Giniatullin, Rashid

2003-01-01

The mode of action of extracellular protons on ATP-gated P2X2 receptors remains controversial as either enhancement or depression of ATP-mediated currents has been reported. By investigating, at different pH, the electrophysiological effect of ATP on P2X2 receptors and complementing it with receptor modelling, the present study suggests a unified mechanism for both potentiation and inactivation of ATP receptors by protons. Our experiments on patch-clamped PC12 cells showed that, on the same cell, mild acidification potentiated currents induced by low ATP concentrations (<0.1 mM) and attenuated responses to high ATP concentrations (>1 mM) with emergence of current fading and rebound. To clarify the nature of the ATP/H+ interaction, we used the Ding and Sachs's “loop” receptor model which best describes the behavior of such receptors with two open states linked via one inactivated state. No effects by protons could be ascribed to H+-mediated open channel block. However, by assuming that protons facilitated binding of ATP to resting as well as open receptors, the model could closely replicate H+-induced potentiation of currents evoked by low ATP doses plus fading and rebound induced by high ATP doses. The latter phenomenon was due to receptor transition to the inactive state. The present data suggest that the high concentration of protons released with ATP (and catecholamines) from secretory vesicles may allow a dual action of H+ on P2X2 receptors. This condition might also occur on P2X2 receptors of central neurons exposed to low pH during ischemia. PMID:12810852

50 Hz hippocampal stimulation in refractory epilepsy: Higher level of basal glutamate predicts greater release of glutamate.

PubMed

Cavus, Idil; Widi, Gabriel A; Duckrow, Robert B; Zaveri, Hitten; Kennard, Jeremy T; Krystal, John; Spencer, Dennis D

2016-02-01

The effect of electrical stimulation on brain glutamate release in humans is unknown. Glutamate is elevated at baseline in the epileptogenic hippocampus of patients with refractory epilepsy, and increases during spontaneous seizures. We examined the effect of 50 Hz stimulation on glutamate release and its relationship to interictal levels in the hippocampus of patients with epilepsy. In addition, we measured basal and stimulated glutamate levels in a subset of these patients where stimulation elicited a seizure. Subjects (n = 10) were patients with medically refractory epilepsy who were undergoing intracranial electroencephalography (EEG) evaluation in an epilepsy monitoring unit. Electrical stimulation (50 Hz) was delivered through implanted hippocampal electrodes (n = 11), and microdialysate samples were collected every 2 min. Basal glutamate, changes in glutamate efflux with stimulation, and the relationships between peak stimulation-associated glutamate concentrations, basal zero-flow levels, and stimulated seizures were examined. Stimulation of epileptic hippocampi in patients with refractory epilepsy caused increases in glutamate efflux (p = 0.005, n = 10), and 4 of ten patients experienced brief stimulated seizures. Stimulation-induced increases in glutamate were not observed during the evoked seizures, but rather were related to the elevation in interictal basal glutamate (R(2) = 0.81, p = 0.001). The evoked-seizure group had lower basal glutamate levels than the no-seizure group (p = 0.04), with no stimulation-induced change in glutamate efflux (p = 0.47, n = 4). Conversely, increased glutamate was observed following stimulation in the no-seizure group (p = 0.005, n = 7). Subjects with an atrophic hippocampus had higher basal glutamate levels (p = 0.03, n = 7) and higher stimulation-induced glutamate efflux. Electrical stimulation of the epileptic hippocampus either increased extracellular glutamate efflux or induced seizures. The magnitude of stimulated

Adaptogens stimulate neuropeptide y and hsp72 expression and release in neuroglia cells.

PubMed

Panossian, Alexander; Wikman, Georg; Kaur, Punit; Asea, Alexzander

2012-01-01

The beneficial stress-protective effect of adaptogens is related to the regulation of homeostasis via mechanisms of action associated with the hypothalamic-pituitary-adrenal axis and the regulation of key mediators of the stress response, such as molecular chaperones, stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cortisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream targets are in response to stimulation by adaptogens. The present study addresses this gap in our knowledge and suggests that an important target for adaptogen mediated stress-protective effector functions is the stress hormone neuropeptide Y (NPY). We demonstrated that ADAPT-232, a fixed combination of adaptogens Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was validated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Taken together our studies suggest that the stimulation and release of the stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress.

Role of hemolysis in red cell adenosine triphosphate release in simulated exercise conditions in vitro.

PubMed

Mairbäurl, Heimo; Ruppe, Florian A; Bärtsch, Peter

2013-10-01

Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

ATP-induced changes in rat skeletal muscle contractility.

PubMed

Gabdrakhmanov, A I; Khayrullin, A E; Grishin, C H; Ziganshin, A U

2015-01-01

Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be

Co-release of noradrenaline and dopamine in the cerebral cortex elicited by single train and repeated train stimulation of the locus coeruleus

PubMed Central

Devoto, Paola; Flore, Giovanna; Saba, Pierluigi; Fà, Mauro; Gessa, Gian Luigi

2005-01-01

Background Previous studies by our group suggest that extracellular dopamine (DA) and noradrenaline (NA) may be co-released from noradrenergic nerve terminals in the cerebral cortex. We recently demonstrated that the concomitant release of DA and NA could be elicited in the cerebral cortex by electrical stimulation of the locus coeruleus (LC). This study analyses the effect of both single train and repeated electrical stimulation of LC on NA and DA release in the medial prefrontal cortex (mPFC), occipital cortex (Occ), and caudate nucleus. To rule out possible stressful effects of electrical stimulation, experiments were performed on chloral hydrate anaesthetised rats. Results Twenty min electrical stimulation of the LC, with burst type pattern of pulses, increased NA and DA both in the mPFC and in the Occ. NA in both cortices and DA in the mPFC returned to baseline within 20 min after the end of the stimulation period, while DA in the Occ reached a maximum increase during 20 min post-stimulation and remained higher than baseline values at 220 min post-stimulation. Local perfusion with tetrodotoxin (TTX, 10 μM) markedly reduced baseline NA and DA in the mPFC and Occ and totally suppressed the effect of electrical stimulation in both areas. A sequence of five 20 min stimulations at 20 min intervals were delivered to the LC. Each stimulus increased NA to the same extent and duration as the first stimulus, whereas DA remained elevated at the time next stimulus was delivered, so that baseline DA progressively increased in the mPFC and Occ to reach about 130 and 200% the initial level, respectively. In the presence of the NA transport (NAT) blocker desipramine (DMI, 100 μM), multiple LC stimulation still increased extracellular NA and DA levels. Electrical stimulation of the LC increased NA levels in the homolateral caudate nucleus, but failed to modify DA level. Conclusion The results confirm and extend that LC stimulation induces a concomitant release of DA and NA

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

PubMed

Uchida, Sae; Kagitani, Fusako

2017-05-12

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

REVIEWS OF TOPICAL PROBLEMS Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor

NASA Astrophysics Data System (ADS)

Romanovsky, Yurii M.; Tikhonov, Alexander N.

2010-12-01

The free energy released upon the enzymatic hydrolysis of adenosine triphosphate (ATP) is the main source of energy for the functioning of the living cell and all multicellular organisms. The overwhelming majority of ATP molecules are formed by proton ATP synthases, which are the smallest macromolecular electric motors in Nature. This paper reviews the modern concepts of the molecular structure and functioning of the proton ATP synthase, and real-time biophysical experiments on the rotation of the 'rotor' of this macromolecular motor. Some mathematical models describing the operation of this nanosized macromolecular machine are described.

Knocking out P2X receptors reduces transmitter secretion in taste buds.

PubMed

Huang, Yijen A; Stone, Leslie M; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E; Kinnamon, Sue C; Roper, Stephen D

2011-09-21

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.

Knocking out P2X receptors reduces transmitter secretion in taste buds

PubMed Central

Huang, Yijen A.; Stone, Leslie M.; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C.; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E.; Kinnamon, Sue C.; Roper, Stephen D.

2011-01-01

In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors (P2X2 and P2X3 double knockout, or “DKO” mice). The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca2+ in taste Receptor (Type II) cells from DKO mice, as from wild type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we employed reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion. PMID:21940456

In Vivo Electrochemical Evidence for Simultaneous 5-HT and Histamine Release in the Rat Substantia Nigra pars Reticulata Following Medial Forebrain Bundle Stimulation

PubMed Central

Hashemi, Parastoo; Dankoski, Elyse C.; Wood, Kevin M.; Ambrose, R. Ellen; Wightman, R. Mark

2011-01-01

Exploring the mechanisms of serotonin (5-hydoxytryptophan (5-HT)) in the brain requires an in vivo method that combines fast temporal resolution with chemical selectivity. Fast-scan cyclic voltammetry (FSCV) is a technique with sufficient temporal and chemical resolution for probing dynamic 5-HT neurotransmission events; however, traditionally it has not been possible to probe in vivo 5-HT mechanisms. Recently, we optimized FSCV for measuring 5-HT release and uptake in vivo in the substantia nigra pars reticulata (SNR) with electrical stimulation of the dorsal raphe nucleus (DRN) in the rat brain. Here, we address technical challenges associated with rat DRN surgery by electrically stimulating 5-HT projections in the medial forebrain bundle (MFB), a more accessible anatomical location. MFB stimulation elicits 5-HT in the SNR; furthermore, we find simultaneous release of an additional species. We use electrochemical and pharmacological methods and describe physiological, anatomical and independent chemical analyses to identify this species as histamine. We also show pharmacologically that increasing the lifetime of extracellular histamine significantly decreases 5-HT release, most likely due to increased activation of histamine H-3 receptors that inhibit 5-HT release. Despite this, under physiological conditions, we find by kinetic comparisons of DRN and MFB stimulations that the simultaneous release of histamine does not interfere with the quantitative 5-HT concentration profile. We therefore present a novel and robust electrical stimulation of the MFB that is technically less challenging than DRN stimulation to study 5-HT and histamine release in the SNR. PMID:21682723

Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

PubMed

Liu, Ying; Geng, Yue-Hang; Yang, Hui; Yang, Han; Zhou, Yan-Ting; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

2018-05-04

Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis. Copyright © 2018. Published by Elsevier B.V.

Beta-phenylethylamine stimulates striatal acetylcholine release through activation of the AMPA glutamatergic pathway.

PubMed

Ishida, Kota; Murata, Mikio; Kato, Masatoshi; Utsunomiya, Iku; Hoshi, Keiko; Taguchi, Kyoji

2005-09-01

Using an in vivo intra-striatal microdialysis technique, we examined the effects of systemically administered beta-phenylethylamine (beta-PEA), a psychomotor stimulating trace amine, on striatal acetylcholine release in freely moving rats. Infusion of N-methyl-D-aspartic acid (NMDA; 10(-5) M) significantly increased acetylcholine release. In addition, locally applied amino-3-hydroxy-5-methylisozasole-4-propionic acid (AMPA; 10(-5) M) significantly increased acetylcholine release in the striatum. Intra-striatal application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M), an AMPA-type glutamatergic receptor antagonist, had little effect on acetylcholine release, while application of MK-801 (10(-5) M, 10(-6) M), an NMDA-type glutamatergic receptor antagonist, significantly reduced acetylcholine release. Acetylcholine within striatal perfusate was significantly increased by intraperitoneal administration of beta-PEA in a dose-dependent manner. This increase in acetylcholine release was completely blocked by application of CNQX (10(-5) M) through the microdialysis probe into the striatum. However, increased acetylcholine response to systemic beta-PEA was unaltered by addition of MK-801 to the perfusion medium. These results suggest a regulatory function of beta-PEA, mediated by AMPA-type glutamatergic receptors, on the release of acetylcholine in the rat striatum.

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.

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. Copyright © 2012 Elsevier Inc. All rights reserved.

The effect of toxins on inorganic phosphate release during actin polymerization.

PubMed

Vig, Andrea; Ohmacht, Róbert; Jámbor, Eva; Bugyi, Beáta; Nyitrai, Miklós; Hild, Gábor

2011-05-01

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

Effects of morphine, physostigmine and raphe nuclei stimulation on 5-hydroxytryptamine release from the cerebral cortex of the cat.

PubMed Central

Aiello-Malmberg, P; Bartolini, A; Bartolini, R; Galli, A

1979-01-01

1. The release of 5-hydroxytryptamine (5-HT) from the cerebral cortex and caudate nucleus of brainstem-transected cats and from the cerebral cortex of rats anaesthetized with urethane was determined by radioenzymatic and biological assay. 2. The stimulation of nucleus linearis intermedius of raphe doubles the basal 5-HT release in the caudate nucleus and increases it 3 fold in the cerebral cortex. The effects of the electrical stimulation of the raphe are potentiated by chlorimipramine. 3. Brain 5-HT release is greatly increased by morphine hydrochloride (6 mg/kg i.v.) and by physostigmine (100 microgram/kg i.v.), but not by DL-DOPA (50 mg/kg i.v.). 4. It is suggested that the 5-HT releasing action of physostigmine can contribute to some of its pharmacological effects such as the analgesic effect so far attributed exclusively to its indirect cholinomimetic activity. 5. The 5-HT releasing action of physostigmine seems unrelated to its anticholinesterase activity. PMID:435680

Protection of ATP-Depleted Cells by Impermeant Strychnine Derivatives

PubMed Central

Dong, Zheng; Venkatachalam, Manjeri A.; Weinberg, Joel M.; Saikumar, Pothana; Patel, Yogendra

2001-01-01

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine. PMID:11238050

Adaptogens Stimulate Neuropeptide Y and Hsp72 Expression and Release in Neuroglia Cells

PubMed Central

Panossian, Alexander; Wikman, Georg; Kaur, Punit; Asea, Alexzander

2011-01-01

The beneficial stress–protective effect of adaptogens is related to the regulation of homeostasis via mechanisms of action associated with the hypothalamic–pituitary–adrenal axis and the regulation of key mediators of the stress response, such as molecular chaperones, stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cortisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream targets are in response to stimulation by adaptogens. The present study addresses this gap in our knowledge and suggests that an important target for adaptogen mediated stress–protective effector functions is the stress hormone neuropeptide Y (NPY). We demonstrated that ADAPT-232, a fixed combination of adaptogens Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was validated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Taken together our studies suggest that the stimulation and release of the stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress. PMID:22347152

Critical Involvement of Extracellular ATP Acting on P2RX7 Purinergic Receptors in Photoreceptor Cell Death

PubMed Central

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-01-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. PMID:21983632

Adding a custom made pressure release valve during air enema for intussusception: A new technique.

PubMed

Ahmed, Hosni Morsi; Ahmed, Osama; Ahmed, Refaat Khodary

2015-01-01

Non-surgical reduction remains the first line treatment of choice for intussusception. The major complication of air enema reduction is bowel perforation. The authors developed a custom made pressure release valve to be added to portable insufflation devices, delivering air at pressures accepted as safe for effective reduction of intussusception in children under fluoroscopic guidance. The aim of this study was to develop a custom made pressure release valve that is suitable for the insufflation devices used for air enema reduction of intussusception and to put this valve into regular clinical practice. An adjustable, custom made pressure release valve was assembled by the authors using readily available components. The valve was coupled to a simple air enema insufflation device. The device was used for the trial of reduction of intussusception in a prospective study that included 132 patients. The success rate for air enema reduction with the new device was 88.2%. The mean pressure required to achieve complete reduction was 100 mmHg. The insufflation pressure never exceeded the preset value (120 mmHg). Of the successful cases, 58.3% were reduced from the first attempt while 36.1% required a second insufflation. Only 5.55% required a third insufflation to complete the reduction. In cases with unsuccessful pneumatic reduction attempt (18.1%), surgical treatment was required. Surgery ranged from simple reduction to resection with a primary end to end anastomosis. No complications from air enema were recorded. The authors recommend adding pressure release valves to ensure safety by avoiding pressure overshoot during the procedure.

Air exposure and sample storage time influence on hydrogen release from tungsten

NASA Astrophysics Data System (ADS)

Moshkunov, K. A.; Schmid, K.; Mayer, M.; Kurnaev, V. A.; Gasparyan, Yu. M.

2010-09-01

In investigations of hydrogen retention in first wall components the influence of the conditions of the implanted target storage prior to analysis and the storage time is often neglected. Therefore we have performed a dedicated set of experiments. The release of hydrogen from samples exposed to ambient air after irradiation was compared to samples kept in vacuum. For air exposed samples significant amounts of HDO and D 2O are detected during TDS. Additional experiments have shown that heavy water is formed by recombination of releasing D and H atoms with O on the W surface. This water formation can alter hydrogen retention results significantly, in particular - for low retention cases. In addition to the influence of ambient air exposure also the influence of storage time in vacuum was investigated. After implantation at 300 K the samples were stored in vacuum for up to 1 week during which the retained amount decreased significantly. The subsequently measured TDS spectra showed that D was lost from both the high and low energy peaks during storage at ambient temperature of ˜300 K. An attempt to simulate this release from both peaks during room temperature storage by TMAP 7 calculations showed that this effect cannot be explained by conventional diffusion/trapping models.

Insulin stimulates synthesis and release of human chorionic gonadotropin by choriocarcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, S.G.; Braunstein, G.D.

1991-03-01

Recent studies have shown that insulin regulates placental lactogen, progesterone, and estrogen production from human trophoblast cells. This study was performed to examine whether insulin also regulates the production of hCG by this type of cell. After 24-36 h of preincubation, JEG-3 and JAR cells (2-3 x 10(5) cells/ml.well) or human term trophoblast cells (1 x 10(6) cells/ml.well) were exposed to the test hormone in serum-free Dulbecco's Modified Eagle's Medium for 24-96 h. Secretion of hCG from JEG-3 cells was stimulated by human insulin, human proinsulin, or porcine insulin in a dose-dependent manner, with lowest effective doses of 6.7, 96,more » and 53 mg/L, respectively. Time-course studies showed that hCG secretion peaked at 72-96 h with insulin exposure; in contrast, no decernable peak was seen without insulin in serum-free media. Exposure of JEG-3 cells for 24 h to 209 mg/liter insulin stimulated hCG synthesis, with 40 +/- 3% more immunoreactive intracellular hCG (P less than 0.05). Cells grown in the presence of insulin and (35S)methionine had 47 +/- 21% more labeled intracellular hCG and 56 +/- 13% more immunoprecipitable (35S)methionine-hCG secreted into the medium than the control cultures (P less than 0.05). During this time period, human placental lactogen release and total trichloroacetice acid-precipitable (35S)methionine protein were not increased. The insulin-induced stimulation of hCG synthesis was inhibited by cycloheximide. Additionally, insulin did not significantly affect total intracellular protein during 24-96 h of incubation. Insulin also increased hCG release from JAR cells, but not from human term trophoblast cells. A mouse monoclonal antibody to the IGF-I receptor inhibited the stimulation of insulin in JEG-3 cells.« less

Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors.

PubMed

zur Nedden, Stephanie; Hawley, Simon; Pentland, Naomi; Hardie, D Grahame; Doney, Alexander S; Frenguelli, Bruno G

2011-04-20

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed Central

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-01-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways. PMID:9276720

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-09-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.

Design and application of an inertial impactor in combination with an ATP bioluminescence detector for in situ rapid estimation of the efficacies of air controlling devices on removal of bioaerosols.

PubMed

Yoon, Ki Young; Park, Chul Woo; Byeon, Jeong Hoon; Hwang, Jungho

2010-03-01

We proposed a rapid method to estimate the efficacies of air controlling devices in situ using ATP bioluminescence in combination with an inertial impactor. The inertial impactor was designed to have 1 mum of cutoff diameter, and its performance was estimated analytically, numerically, and experimentally. The proposed method was characterized using Staphylococcus epidermidis, which was aerosolized with a nebulizer. The bioaerosol concentrations were estimated within 25 min using the proposed method without a culturing process, which requires several days for colony formation. A linear relationship was obtained between the results of the proposed ATP method (RLU/m(3)) and the conventional culture-based method (CFU/m(3)), with R(2) 0.9283. The proposed method was applied to estimate the concentration of indoor bioaerosols, which were identified as a mixture of various microbial species including bacteria, fungi, and actinomycetes, in an occupational indoor environment, controlled by mechanical ventilation and an air cleaner. Consequently, the proposed method showed a linearity with the culture-based method for indoor bioaerosols with R(2) 0.8189, even though various kinds of microorganisms existed in the indoor air. The proposed method may be effective in monitoring the changes of relative concentration of indoor bioaerosols and estimating the effectiveness of air control devices in indoor environments.

Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors

PubMed Central

de Souza, Devandir Antonio; Borges, Antonio Carlos; Santana, Ana Carolina; Oliver, Constance; Jamur, Maria Célia

2015-01-01

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization. PMID:26633538

Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B.

PubMed

Tadini-Buoninsegni, Francesco; Smeazzetto, Serena

2017-04-01

ATP7A and ATP7B are Cu + -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu + ions across the membrane from delivery to acceptor proteins without establishing a free Cu + gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca 2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper. © 2016 IUBMB Life, 69(4):218-225, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Exercise in cold air and hydrogen peroxide release in exhaled breath condensate.

PubMed

Marek, E; Volke, J; Mückenhoff, K; Platen, P; Marek, W

2013-01-01

Athletes have changes in the lung epithelial cells caused by inhalation of cold and dry air. The exhaled breath condensate contains a number of mediators from the respiratory system and H(2)O(2) is described as a marker of airways inflammation. The aim of this study was to determine the influence of exercise combined with cold air on the H(2)O(2) release in the exhaled breath. Twelve males (23.1 ± 1.5 years) were randomly assigned at 2 different days (1 day rest) to perform a 50 min run (75-80% of their max. heart rate) under normal (N) laboratory (18.1 ± 1.1°C) or cold (C) field condition (-15.2 ± 3.1°C). Before and immediately after each run, the EBC was collected under laboratory conditions and was analyzed amperometrically. Prior to the two runs, H(2)O(2) concentrations were 145.0 ± 31.0 (N) and 160.0 ± 49.1 nmol/L (C) and theoretical release was 70.3 ± 37.1 (N) and 82.6 ± 27.1 pmol/min (C) (p > 0.05). After each run, H(2)O(2) concentration increased significantly to 388.0 ± 22.8 nmol/L (N) and 622.1 ± 44.2 nmol/L (C) (p < 0.05), along with an increase in the theoretical release: 249.2 ± 35.7 pmol/min (N) and 400.9 ± 35.7 pmol/min (C) (p < 0.05). We conclude that release of H(2)O(2) into the EBC takes place under both resting conditions and after exercise. The concentration and release of H(2)O(2) increased after exercise in cold air compared to resting and laboratory conditions, which points to an increase in inflammatory and oxidative stress.

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

PubMed

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

2015-01-15

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

Landfill-stimulated iron reduction and arsenic release at the Coakley Superfund Site (NH).

PubMed

deLemos, Jamie L; Bostick, Benjamin C; Renshaw, Carl E; Stürup, Stefan; Feng, Xiahong

2006-01-01

Arsenic is a contaminant at more than one-third of all Superfund Sites in the United States. Frequently this contamination appearsto resultfrom geochemical processes rather than the presence of a well-defined arsenic source. Here we examine the geochemical processes that regulate arsenic levels at the Coakley Landfill Superfund Site (NH), a site contaminated with As, Cr, Pb, Ni, Zn, and aromatic hydrocarbons. Long-term field observations indicate that the concentrations of most of these contaminants have diminished as a result of treatment by monitored natural attenuation begun in 1998; however, dissolved arsenic levels increased modestly over the same interval. We attribute this increase to the reductive release of arsenic associated with poorly crystalline iron hydroxides within a glaciomarine clay layer within the overburden underlying the former landfill. Anaerobic batch incubations that stimulated iron reduction in the glaciomarine clay released appreciable dissolved arsenic and iron. Field observations also suggest that iron reduction associated with biodegradation of organic waste are partly responsible for arsenic release; over the five-year study period since a cap was emplaced to prevent water flow through the site, decreases in groundwater dissolved benzene concentrations at the landfill are correlated with increases in dissolved arsenic concentrations, consistent with the microbial decomposition of both benzene and other organics, and reduction of arsenic-bearing iron oxides. Treatment of contaminated groundwater increasingly is based on stimulating natural biogeochemical processes to degrade the contaminants. These results indicate that reducing environments created within organic contaminant plumes may release arsenic. In fact, the strong correlation (>80%) between elevated arsenic levels and organic contamination in groundwater systems at Superfund Sites across the United States suggests that arsenic contamination caused by natural degradation of

The role of Ca2+ signaling in the coordination of mitochondrial ATP production with cardiac work

PubMed Central

Balaban, Robert S.

2009-01-01

The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca2+. Since Ca2+ initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca2+ is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca2+ gradient near equilibrium with the free energy of ATP, cytosolic Ca2+ release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca2+ can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca2+ with work in numerous preparations as well as the localization of mitochondria near Ca2+ release sites. How cytosolic Ca2+ signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca2+ sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca2+ apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart. PMID:19481532

Hierarchical drug release of pH-sensitive liposomes encapsulating aqueous two phase system.

PubMed

Zhang, Xunan; Zong, Wei; Bi, Hongmei; Zhao, Kunming; Fuhs, Thomas; Hu, Ying; Cheng, Wenlong; Han, Xiaojun

2018-06-01

As promising drug delivery vehicles, previous investigations of liposomes as carriers are primarily focused on insertion and modification of lipid membrane interfaces. The utility of the inner core seems to be overlooked. Herein, we developed pH-sensitive liposomes (PSLs) containing an aqueous two phase system (ATPS), and intriguingly discovered their hierarchical release under acidic stimuli. ATPS containing two polymers (poly(ethylene glycol) (PEG) and dextran) is homogeneous above phase transition temperature when producing ATPS-liposomes, and separated into PEG-rich phase and dextran-rich phase after cooling down to room temperature. The overall release time of ATPS-liposomes is divided into two stages and prolonged compared to simple aqueous liposomes. The unique release profile is due to the disproportional distribution of drugs in two phases. Doxorubicin (DOX) is loaded in the ATPS-liposomes, and their half maximum inhibition concentration on HeLa cells is 0.018 μmol L -1 , which means 27.5 fold increase in inhibition efficiency over free DOX. Copyright © 2018 Elsevier B.V. All rights reserved.