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Diabetes mellitus adversely affects human bones and increases the risk of developing osteoporosis. In the present study, treatment with 30 mmol/l glucose was used to establish a high glucose (HG) cell model in vitro. Plasmids were used to overexpress the P2X purinoceptor 7 (P2X7) gene. Brilliant blue G and (4‑benzoyl‑benzoyl)‑ATP were used as a P2X7 antagonist and agonist, respectively. Proliferation of osteogenic MC3T3‑E1 cells and alkaline phosphatase (ALP) activity were determined using MTT and colorimetric assays, respectively. Alizarin Red S was used to assess calcification of MC3T3‑E1 cells. Western blotting and reverse transcription‑quantitative PCR were performed to determine protein and mRNA expression levels. The results demonstrated that HG inhibited MC3T3‑E1 cell proliferation and P2X7 expression, reduced calcification, and downregulated the expression levels of ALP and osteocalcin (Ocn) in MC3T3‑E1 cells. Overexpression of P2X7 in HG conditions increased calcification and proliferation, and upregulated the levels of ALP and Ocn in MC3T3‑E1 cells. Inhibition of P2X7 downregulated the expressions of ALP and Ocn in MC3T3‑E1 cells under HG conditions. Therefore, the present results indicated that HG caused damage to osteogenic MC3T3‑E1 cells. Thus, P2X7 may be a regulatory factor that may be used to counteract the effects of HG on osteogenesis.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E 2 (PGE 2 ) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE 2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE 2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE 2 -triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE 2 on MCs may be involved in Ag-independent hypersensitivity in vivo ., Competing Interests: The authors declare no conflict of interest.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Brain tissue manganese (Mn) accumulation is a cirrhosis-associated complication. Cellular mitochondria are among the potential targets for Mn-induced cytotoxicity. Taurine is one of the most abundant amino acids with high concentrations in human brain tissue. Several pharmacological properties including regulation of mitochondrial function are attributed to taurine. The current investigation was designed to evaluate the effect of taurine on Mn-induced mitochondrial impairment in isolated mice brain mitochondria. The brain mitochondria were exposed to increasing concentrations of Mn (0.1-10 mM). Taurine (0.1, 1, and 10 mM) was added as the protective agent. The severe collapse of mitochondrial membrane potential, decreased mitochondrial dehydrogenases activity, mitochondrial swelling, and depleted mitochondrial adenosine triphosphate (ATP) were evident in Mn-exposed mitochondria. It was found that taurine administration preserved mitochondrial ATP, prevented mitochondrial depolarization and swelling, and increased mitochondrial dehydrogenases activity. These data suggest mitochondrial protection as an underlying mechanism for the protective effects of taurine against Mn toxicity., (© 2018 Wiley Periodicals, Inc.)

Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 44(3):245-262, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria., (Copyright © 2017. Published by Elsevier B.V.)

Mitochondrial dysfunction is elevated in very early stages of Alzheimer's disease and exacerbates oxidative stress, which contributes to disease pathology. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APP SWE and PSEN1 dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter (SVCT2 +/- ) and transgenic APP/PSEN1 mice with heterozygous SVCT2 expression. Mitochondrial isolates from SVCT2 +/- mice were observed to consume less oxygen using high-resolution respirometry, and also exhibited decreased mitochondrial membrane potential compared to wild type isolates. Conversely, isolates from young (4 months) APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were also produced in mitochondria isolated from both APP/PSEN1 and SVCT2 +/- mice compared to wild type isolates. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that both presence of amyloid and ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early, prodromal stage of Alzheimer's disease, although occurring via different pathways. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease, particularly in populations most at risk for Alzheimer's disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Functional perturbation of mitochondria is associated with fulminant hepatic failure (FHF). d-Galactosamine/lipopolysaccharide (d-GalN/LPS)-induced FHF is a renowned model to evaluate the efficacy of hepatoprotective agents. Lycopene is an antioxidant and phytonutrient from the carotenoid family. The health benefits of lycopene are prominent against cancer and cardiovascular, lung, liver, and skin problems. Recent studies have demonstrated the hepatoprotective, antidyslipidemic, and antioxidant roles of lycopene. The current study was designed to appraise the ability of lycopene to prevent mitochondrial dysfunction during the d-GalN/LPS-induced FHF. The administration of d-GalN/LPS (300 mg and 30 μg/kg body weight, respectively) to the experimental rats induced several disturbances in mitochondrial function. The lipid peroxide and hydrogen peroxide levels were increased (p < 0.05). The activities of mitochondrial antioxidants, tricarboxylic acid (TCA) cycle, and electron transport chain enzymes and the cellular adenosine triphosphate (ATP) content were decreased (p < 0.05). Lycopene (10 mg/kg body weight for 6 days) pretreatment attenuated lipid peroxidation and prohibited the excessive synthesis of hydrogen peroxide. The d-GalN/LPS-induced impairment in ATP production and increased enzyme activities were effectively prevented by the lycopene administration. The lycopene-mediated mitochondrial protection was mainly ascribed to the strong antioxidant potential of this phytonutrient. Molecular modeling results obtained show evidence that lycopene inhibits several lipoxygenases and provides rationale for the observed prevention of lipid peroxidation in the mitochondrial membrane. The carotenoid lycopene combatted oxidative stress, scavenged free radicals, prevented ROS generation, and inhibited the toxic effects of d-GalN/LPS during FHF.

Cardiac ischemia and reperfusion (I/R) injury remains a challenge for clinicians. Ginsenoside Rb1 (Rb1) has been reported to have the ability to attenuate I/R injury, but its effect on energy metabolism during cardiac I/R and the underlying mechanism remain unknown. In this study, we detected the effect of Rb1 on rat myocardial blood flow, myocardial infarct size, cardiac function, velocity of venule red blood cell, myocardial structure and apoptosis, energy metabolism and change in RhoA signaling pathway during cardiac I/R injury. In addition, the binding affinity of RhoA to Rb1 was detected using surface plasmon resonance (SPR). Results showed that Rb1 treatment at 5 mg/kg/h protected all the cardiac injuries induced by I/R, including damaged myocardial structure, decrease in myocardial blood flow, impaired heart function and microcirculation, cardiomyocyte apoptosis, myocardial infarction and release of myocardial cTnI. Rb1 also inhibited the activation of RhoA signaling pathway and restored the production of ATP during cardiac I/R. Moreover, SPR assay showed that Rb1 was able to bind to RhoA in a dose-dependent manner. These results indicate that Rb1 may prevent I/R-induced cardiac injury by regulation of RhoA signaling pathway, and may serve as a potential regime to improve percutaneous coronary intervention outcome.

Global pollination is threatened by declining insect pollinator populations that may be linked to neonicotinoid pesticide use. Neonicotinoids over stimulate neurons and depolarize their mitochondria, producing immobility and death. However, mitochondrial function can be improved by near infrared light absorbed by cytochrome c oxidase in mitochondrial respiration. In flies, daily exposure to 670nm light throughout life increases average lifespan and aged mobility, and reduces systemic inflammation. Here we treat bumble bees with Imidacloprid a common neonicotinoid. This undermined ATP and rapidly induced immobility and reduced visual function and survival. Bees exposed to insecticide and daily to 670nm light showed corrected ATP levels and significantly improved mobility allowing them to feed. Physiological recordings from eyes revealed that light exposure corrected deficits induced by the pesticide. Overall, death rates in bees exposed to insecticide but also given 670nm light were indistinguishable from controls. When Imidacloprid and light exposure were withdrawn, survival was maintained. Bees and insects generally cannot see deep red light so it does not disturb their behaviour. Hence, we show that deep red light exposure that improves mitochondrial function, reverses the sensory and motor deficits induced by Imidacloprid. These results may have important implications as light delivery is economic and can be placed in hives/colonies., Competing Interests: The authors have declared that no competing interests exist.

Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2'-deoxy ATP, 3'-deoxy ATP, Ap5A and αβmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2' hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αβmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs.

Coenzyme Q (CoQ, or ubiquinone) is a remarkable lipid that plays an essential role in mitochondria as an electron shuttle between complexes I and II of the respiratory chain, and complex III. It is also a cofactor of other dehydrogenases, a modulator of the permeability transition pore and an essential antioxidant. CoQ is synthesized in mitochondria by a set of at least 12 proteins that form a multiprotein complex. The exact composition of this complex is still unclear. Most of the genes involved in CoQ biosynthesis (COQ genes) have been studied in yeast and have mammalian orthologues. Some of them encode enzymes involved in the modification of the quinone ring of CoQ, but for others the precise function is unknown. Two genes appear to have a regulatory role: COQ8 (and its human counterparts ADCK3 and ADCK4) encodes a putative kinase, while PTC7 encodes a phosphatase required for the activation of Coq7. Mutations in human COQ genes cause primary CoQ(10) deficiency, a clinically heterogeneous mitochondrial disorder with onset from birth to the seventh decade, and with clinical manifestation ranging from fatal multisystem disorders, to isolated encephalopathy or nephropathy. The pathogenesis of CoQ(10) deficiency involves deficient ATP production and excessive ROS formation, but possibly other aspects of CoQ(10) function are implicated. CoQ(10) deficiency is unique among mitochondrial disorders since an effective treatment is available. Many patients respond to oral CoQ(10) supplementation. Nevertheless, treatment is still problematic because of the low bioavailability of the compound, and novel pharmacological approaches are currently being investigated. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi., (Copyright © 2016. Published by Elsevier B.V.)

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival., (© 2016 Society for Laboratory Automation and Screening.)

Successful clinical outcomes from transplantation of hematopoietic stem cells (HSCs) depend upon efficient HSC homing to bone marrow (BM), subsequent engraftment, and, finally, BM repopulation. Homing of intravenously administered HSCs from peripheral blood (PB) through the circulation to the BM stem cell niches, which is the first critical step that precedes their engraftment, is enforced by chemotactic factors released in the BM microenvironment that chemoattract HSCs. These chemotactic factors include α-chemokine stromal-derived factor 1 (SDF-1), the bioactive phosphosphingolipids sphingosine-1-phosphate (S1P) and ceramid-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. Stem cells may also respond to a Ca(2+) or H(+) gradient by employing calcium- or proton-sensing receptors, respectively. In this review, we will present emerging strategies based on ex vivo manipulation of graft HSCs that are aimed at enhancing the responsiveness of HSCs to BM-secreted chemoattractants and/or promoting HSC adhesion and seeding efficiency in the BM microenvironment.

Malonate, a classic inhibitor of respiratory electron transport chain, induces mitochondrial stress. Stress granules (SGs) are a kind of dynamic foci structure during stress. The study on the connection of mitochondrial stress and SG assembly is still limited. Here, we demonstrated that malonate treatment leads to SG formation and translation inhibition, apart from mitochondrial stress, including enhanced ROS formation, reduced mitochondrial Δψm and ATP level. The phosphorylation levels of eIF2α and 4EBP1 protein were affected upon mitochondrial dysfunction. However, knockdown of 4EBP1 affected SG formation, rather than eIF2α. In addition, an increase of ATP level under mitochondrial stress enhanced malonate-induced SG aggregation. Overall, malonate stimulation triggers mitochondrial stress and induces the assembly of non-canonical cellular SGs via 4EBP1 pathway., (© 2015 Federation of European Biochemical Societies.)

Low-level laser (light) therapy has been used before exercise to increase muscle performance in both experimental animals and in humans. However, uncertainty exists concerning the optimum time to apply the light before exercise. The mechanism of action is thought to be stimulation of mitochondrial respiration in muscles, and to increase adenosine triphosphate (ATP) needed to perform exercise. The goal of this study was to investigate the time course of the increases in mitochondrial membrane potential (MMP) and ATP in myotubes formed from C2C12 mouse muscle cells and exposed to light-emitting diode therapy (LEDT). LEDT employed a cluster of LEDs with 20 red (630 ± 10 nm, 25 mW) and 20 near-infrared (850 ± 10 nm, 50 mW) delivering 28 mW cm(2) for 90 s (2.5 J cm(2)) with analysis at 5 min, 3 h, 6 h and 24 h post-LEDT. LEDT-6 h had the highest MMP, followed by LEDT-3 h, LEDT-24 h, LEDT-5 min and Control with significant differences. The same order (6 h > 3 h > 24 h > 5 min > Control) was found for ATP with significant differences. A good correlation was found (r = 0.89) between MMP and ATP. These data suggest an optimum time window of 3-6 h for LEDT stimulate muscle cells., (© 2014 The American Society of Photobiology.)

Neuroprotection may prevent or forestall the progression of incurable eye diseases, such as retinitis pigmentosa, one of the major causes of adult blindness. Decreased cellular ATP levels may contribute to the pathology of this eye disease and other neurodegenerative diseases. Here we describe small compounds (Kyoto University Substances, KUSs) that were developed to inhibit the ATPase activity of VCP (valosin-containing protein), the most abundant soluble ATPase in the cell. Surprisingly, KUSs did not significantly impair reported cellular functions of VCP but nonetheless suppressed the VCP-dependent decrease of cellular ATP levels. Moreover, KUSs, as well as exogenous ATP or ATP-producing compounds, e.g. methylpyruvate, suppressed endoplasmic reticulum stress, and demonstrably protected various types of cultured cells from death, including several types of retinal neuronal cells. We then examined their in vivo efficacies in rd10, a mouse model of retinitis pigmentosa. KUSs prevented photoreceptor cell death and preserved visual function. These results reveal an unexpected, crucial role of ATP consumption by VCP in determining cell fate in this pathological context, and point to a promising new neuroprotective strategy for currently incurable retinitis pigmentosa.

Hypothermia is a key symptom of sepsis, but the mechanism(s) leading to hypothermia during sepsis is largely unknown and thus no effective therapy is available for hypothermia. Therefore, it is important to investigate the mechanism and develop effective therapeutic methods. Lipopolysaccharide (LPS)-induced hypothermia accompanied by excess nitric oxide (NO) production leads to a reduction in energy production in wild-type mice. However, mice lacking inducible nitric oxide synthase did not suffer from LPS-induced hypothermia, suggesting that hypothermia is associated with excess NO production during sepsis. This observation is supported by the treatment of wild-type mice with α-lipoic acid (LA) in that it effectively attenuates LPS-induced hypothermia with decreased NO production. We also found that LA partially restored ATP production, and activities of the mitochondrial enzymes involved in energy metabolism, which were inhibited during sepsis. These data suggest that hypothermia is related to mitochondrial dysfunction, which is probably compromised by excess NO production and that LA administration attenuates hypothermia mainly by protecting mitochondrial enzymes from NO damage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: The functional significance of purinergic P2 receptors in the myometrium is unclear. We previously reported the ATP-induced ionic currents in rat myometrial cells, causing uterine contractility. The aim of this study is to determine the main P2X receptors that carry the adenosine triphosphate (ATP)-induced currents., Study Design: We cloned predominantly expressed P2X7 receptors from rat myometrium and transfected into cultured COS-7 cells. Reconstructed P2X7 receptor currents were characterized using the whole-cell patch clamp method., Results: Extracellular ATP induced currents through P2X7 receptors with effective concentration (EC(50)) of 155 μmol/L, without desensitization. The myometrial P2X7 receptor was permeable to multiple monovalent cations with conductances ranked as K(+)>Cs(+)>Li(+)>Na(+). It was activated by P2X receptor agonists, with effectiveness ranked as 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP)>>ATP>αβ-methylene ATP (αβ-MeATP)>2-methylthio ATP (2-MeSATP)>uridine triphosphate (UTP)>guanosine triphosphate (GTP)>adenosine diphosphate (ADP). These currents were blocked by selective P2X7 receptor antagonists and extracellular Mg(2+)., Conclusion: P2X7 receptors carry ATP-induced currents in rat myometrial cells.

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

ATP-induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC(50) of 50 μm in somata versus 9.6 μm in terminals). The ATP-induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC(50) of 3.6 μm in somata versus 11.6 μm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP-γ-S and benzoyl-benzoyl-ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC(50) , inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 Blackwell Publishing Ltd.)

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50 μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Memory consolidation in a discriminative bead pecking task is modulated by endogenous adenosine triphosphate (ATP) acting at purinergic receptors in the hippocampus. Consolidation, from short- to intermediate- to long-term memory during two distinct periods following training, was blocked by the non-selective P2 purinergic receptor antagonist PPADS (pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) tetrasodium salt hydrate and the specific P2Y1 receptor antagonist MRS2179. Direct injections of the ATP agonists (ATPγS and ADPβS) potentiated memory consolidation and the effect of ADPβS was blocked by MRS2179, suggesting an important role of ATP on memory consolidation via the P2Y1 receptor in the chick hippocampus. Incubation of astrocytes with ATPγS and ADPβS resulted in the increase of intracellular calcium ([Ca2+]i), the latter being blocked by MRS2179 suggesting a specific role for P2Y1 receptors in the calcium response. This response was prevented by blocking astrocytic oxidative metabolism with fluoroacetate. We argue that the source of the ATP acting on neuronal P2Y1 receptors is most likely to be astrocytes. Thrombin selectively increases [Ca2+]i in astrocytes but not in neurones. The main findings of the present study are: (a) astrocytic [Ca2+]i plays an important role in the consolidation of short-term to long-term memory; and (b) ATP released from chick astrocytes during learning modulates neuronal activity through astrocytic P2Y1 receptors.

Research of efficiency and safety of Vasonat has been carried out. 31 patients aged 46-75 years who had had hemispheric athero- thrombotic or hemodynamic schemic stroke with moderate severity and being treated in 2-5-month of early rehabilitation period have been observed. The control group of patients received placebo. Results of the study, 4 weeks treatment using Vasonat in the dosage of 500 mg/day have shown positive effect on common signs of the disease by decreasing headache intensity, dizziness, a nausea, and on focal neurological symptoms by decreasing hemiparesis degree; psychoemotional and mnestic activity (main memory and attention) improved as well. It was more distinct in patients with localization of the stroke in the right hemisphere of the brain. It was also noted more rapid improvement of motion function and quality of life of patients.

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.

The effects of P2-receptor agonists on myocardial contractility were examined in rats aging 14-100 days. ATP and its stable analog 2-methylthio-ATP potentiated the contraction force of isolated myocardial strips in a dose-dependent manner. The agonist concentrations producing the positive inotropic effect increased from days 14 to 100 of life. The efficiency of extracellular purines depends on animal age: in 14- and 56-day rats the positive inotropic effects of ATP and 2-methylthio-ATP were similar, while in 100-day rats ATP produced more pronounced effect than 2-methylthio-ATP.

Background: The release of adenosine triphosphate (ATP) from the airway epithelial cells during the inflammatory process is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease., Objective: This study was designed to determine whether extracellular ATP is involved in the bronchial hyperresponsiveness as an interaction between epithelium and smooth muscle in the airways., Methods: We examined the contractile response to methacholine (MCh) before and after exposure to low concentrations (< or = 10 microm) of ATP in isolated, epithelium-denuded guinea-pig tracheal smooth muscle by measuring isometric tension. Intracellular Ca2+ concentrations ([Ca2+]i) were assessed by fluorescent intensities of fura-2., Results: MCh-induced contractile force was increased with no change in [Ca2+]i after exposure to 10 microm ATP for 15 min. The ability of ATP to enhance the MCh-induced contraction was markedly attenuated by suramin, a non-selective P2 receptor inhibitor. Pre-incubation with ATPgammaS, a non-hydrolysable analogue of ATP and alpha,beta-meATP, a P2X agonist, also enhanced the MCh-induced contraction. In contrast, uracil triphosphate, a P2Y agonist, did not affect the MCh-induced contraction. Y-27632, a Rho-kinase inhibitor, suppressed the ability of ATP to enhance the MCh-induced contraction. Moreover, PP1 and PP2, Src tyrosin kinase inhibitors, suppressed the enhancement of MCh-induced contraction by ATP., Conclusion: Pre-treatment with ATP induces hyperresponsiveness to MCh mediated by Ca2+ sensitization via the P2X receptor in airway smooth muscle. The present findings suggest the possible involvement of both the Rho-kinase and Src pathways in the intracellular mechanism of this phenomenon.

This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [(3)H]thymidine/5-bromo-2'-deoxyuridine incorporation and the number of cells in both a time- and dose-dependent manner. AMP-CPP (a P2X(1) and P2X(3) agonist), ATP-gammaS (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [(3)H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X(3), P2X(4), P2Y(1), and P2Y(2) expression in mouse ESCs. Adenylate cyclase inhibitor (SQ 22536), phospholipase C inhibitors (neomycin or U 73122), and protein kinase C (PKC) inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC alpha, delta, and zeta from the cytosol to the membrane compartment. ATP and its agonists increased [Ca(2+)](i). In addition, the ATP-induced increase in [(3)H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca(2+) chelator) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (intracellular Ca(2+) chelator). ATP phosphorylated Akt and p44/42 mitogen-activated protein kinases (MAPKs) in a time-dependent manner, and either suramin or reactive blue 2 (RB2) blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [(3)H]thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and the MAPK kinase inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 levels. In conclusion, ATP stimulates mouse ESC proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.

In the present study, ATP-activated currents (I(ATP)s) recorded from rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique are classified into three types (F, I and S) based on the characteristics of their activation and desensitization. The time of rising phase (R(10-90)) of types F, I and S of I(ATP) is measured to be 33.6+/-4.5, 62.2+/-9.9 and 302.1+/-62.0 ms respectively, and positively correlated to cell size. The time of decaying phases (D(10-90)) of types F and S is 399.4+/-58.2 and >1500 ms, respectively. The dose-response curves for the three types of I(ATP) show that their EC(50) values are close (3.44 x 10(-5), 4.89 x 10(-5) and 4.14 x 10(-5) M for types F, I and S respectively, P>0.05). Their reversal potentials are basically the same, varying from +4 to +10 mV. In addition, using whole-cell patch clamp technique in combination with single cell immunohistochemical staining for P2X receptor subunits, our results suggest that the type distinction of ATP-activated current was associated with cell size and P2X receptor subunits: small-sized cells with type F of I(ATP) express only P2X1 and/or P2X3 subunits, while cells with types S and I of I(ATP) express P2X2 or P2X4 in addition to P2X1 and P2X3.

Recently, secretory granule Ca(2+) storage protein chromogranin B (CGB) was shown to be present in the nucleoplasm proper in a complex structure that consists of the inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and the phospholipids. Further, the amounts of IP(3)Rs present in the nucleus of bovine chromaffin cells were shown to be comparable to that of the endoplasmic reticulum. Therefore, we investigated here the potential contribution of nuclear CGB on the IP(3)-dependent Ca(2+) mobilization in the nucleus, using both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells. Chromogranin A (CGA) expression in the NIH3T3 cells, which do not contain intrinsic chromogranins, increased the IP(3)-induced Ca(2+) releases in the nucleus by 45%, while CGB expression in the same cells increased the IP(3)-induced Ca(2+) releases in the nucleus by 80%. Microinjection of IP(3) into the nucleus of CGB-expressing NIH3T3 cells increased the IP(3)-dependent nuclear Ca(2+) mobilization approximately 3-fold, whereas in CGA-expressing cells it remained the same as that of control cells. In contrast, inhibition of CGA expression in PC12 cells by siRNA treatment decreased the IP(3)-induced Ca(2+) releases in the nucleus by 17%, while inhibition of CGB expression decreased the IP(3)-induced Ca(2+) releases in the nucleus by 55%. Microinjection of IP(3) into the nucleus of siCGB-treated PC12 cells decreased the IP(3)-dependent nuclear Ca(2+) mobilization by approximately 75%, whereas in siCGA-treated cells it remained the same as that of control cells. Given the presence of CGB in the nucleus, these results further highlight the critical contribution of nuclear CGB in the IP(3)-induced Ca(2+) release in the nucleus.

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca(2+)](i)) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X(6) receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X(1-5) and P2X(7)) and G-protein-coupled P2Y receptors (i.e., P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12)). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y(14) receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca(2+)](i). While ATP induced rapid and transient [Ca(2+)](i) increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2'-4'-disulfonic acid and oxidized ATP), the P2X(1)/P2X(3) agonist alphabetameATP produced no changes. Conversely, the P2X(7) agonist BzATP markedly increased [Ca(2+)](i); the presence and function of the P2X(7) receptor was also confirmed by the formation of the P2X(7) pore. ADP and 2meSADP also produced [Ca(2+)](i) increases antagonized by the P2Y(1) antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y(14) receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X(7) and P2Y(1) receptors; additional receptors (i.e., P2X(2), P2X(4), P2X(5), P2Y(2), P2Y(4), and P2Y(14)) may also contribute., (Copyright 2003 Wiley-Liss, Inc.)

1. Although pharmacological preconditioning (PPC) has emerged as an alternative to ischaemic preconditioning (IPC) in cardioprotection, the efficacy of PPC compared with IPC has not been investigated. Because IPC is mediated by complex signalling cascades arising from multiple triggers, we have hypothesized that combined PPC is necessary to mimic IPC. 2. Isolated and perfused rat hearts underwent IPC by three cycles of 5 min ischaemia and 5 min reperfusion before 30 min global ischaemia followed by 120 min reperfusion. Adenosine (30 micromol/L), diazoxide (50 micromol/L) and s-nitroso-N-acetylpenicillamine (SNAP; 50 micromol/L) were added for 25 min just before (pretreatment modality) or 45 min before (PPC modality) the index ischaemia. 3. Ischaemic preconditioning significantly improved isovolumic left ventricular (LV) function and reduced infarct size. Although pretreatment with adenosine, diazoxide or SNAP alone was capable of reducing infarct size, PPC with each drug alone or in a combination of two drugs except for diazoxide plus SNAP failed to reduce infarct size. In contrast, PPC in combination with adenosine, diazoxide and SNAP (triple combination PPC) conferred significant improvement of LV function and reduction of infarct size that was as effective as IPC. 4. Cardioprotection afforded by triple combination PPC was abolished by the Gi/o-protein inhibitor pertussis toxin, the mitochondiral KATP channel inhibitor 5-hydroxydecanoate or the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). 5. Protein kinase C (PKC)-epsilon in the particulate fraction was activated throughout preconditioning ischaemia and reperfusion. Although PKC-epsilon was activated during treatment with adenosine, diazoxide or SNAP alone, it was inactivated after washout. In contrast, PKC-epsilon remained activated after triple combination PPC. The PKC inhibitor chelerythrine abolished activation of PKC-epsilon and cardioprotection afforded by IPC and triple combination PPC. 6. These results demonstrate that combined PPC with a G-protein-coupled receptor agonist, a mitochondrial KATP channel opener and an NO donor is necessary to mimic IPC and such synergistic cardioprotection is associated with enhanced and sustained activation of PKC-epsilon.

Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10-300 microM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P(2)y receptor agonists (ADPbetaS and 2-methylthio-ATP) on LPS-induced TNF-alpha release. The rank order of inhibitory potency of endogenous purines on TNF-alpha release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 microM 2-methylthio-ATP showed a potency similar to 100 microM ATP. The addition of ADPbetaS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P(2)y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.

In primary sensory neurons, extracellular ATP plays important roles in nociception and afferent neurotransmission. Here we investigated the effects of ATP on axonal transport in cultured adult mouse dorsal root ganglion neurons using video-enhanced microscopy. Continuous application (26 min) of ATP (100 microM) significantly increased axonal transport of membrane-bound organelles in anterograde and retrograde directions. All neurons tested (n=5) responded to ATP. The number of transported organelles per min began to increase within 2 min and peaked at 11-14 min after the start of ATP application, and thereafter gradually declined. The peak values in both directions were approximately 140% of the initial values before application. The P2 receptor antagonist suramin (1 mM) completely blocked the effect of ATP. Uridine 5'-triphosphate (UTP; 100 microM) produced a similar effect to ATP, with peak values at 11 min reaching 140% in both directions (n=6). ADP (100 microM; n=5), alpha,beta-methylene ATP (100 microM; n=6), or 2-methylthio ATP (100 microM; n=5) had no effect on axonal transport. Our findings indicate that extracellular ATP is able to increase axonal transport in primary sensory neurons. The equal potency of ATP and UTP with no detectable response to ADP, alpha,beta-methylene ATP, or 2-methylthio ATP suggests the possible involvement of P2Y(2) receptors. Extracellular ATP may play an important role in the modulation of axonal transport in sensory neurons.

There is strong evidence for the presence of nucleotide (P2) receptors in sensory neurons, which might play a role in the transmission of pain signals. In contrast, virtually nothing is known about P2 receptors in satellite glial cells (SGCs), which are the main glial cells in sensory ganglia. We investigated the possibility that P2 receptors exist in SGCs in murine trigeminal ganglia, using Ca(2+) imaging, patch-clamp recordings, and immunohistochemistry. We found that ATP caused an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in SGCs. As adenosine had no effect on [Ca(2+)](i), and the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid largely blocked the response to ATP we conclude that P1 receptors did not contribute to the responses. We obtained the following evidence that the responses to ATP were mediated by metabotropic P2Y receptors: (i) persistence of the responses in Ca(2+)-free solution, (ii) inhibition of the response by cyclopiazonic acid, (iii) [Ca(2+)](i) increases in response to the P2Y agonists uridine triphosphate, adenosine thiodiphosphate, and 2-methylthio ADP, and (iv) failure of the P2X agonist alpha,beta-methylene ATP to elicit a response. Agonists of P2Y(1) receptors and uridine triphosphate, an agonist at P2Y(2) and P2Y(4) receptors, induced [Ca(2+)](i) increases suggesting that at least these P2Y receptor subtypes are present on SGCs. Using an antibody against the P2Y(4) receptor, we found immunopositive SGCs. Patch-clamp recordings of SGCs did not reveal any inward current due to ATP. Therefore, there was no evidence for the activation of ionotropic P2X receptors under the present conditions. The results indicate the presence of functional nucleotide (P2Y) receptors in SGCs.

Succinate-dependent Ca2+ accumulation was investigated in rat liver mitochondria and in operation biopsies of patients under states either with prevalence of adrenergic influences (administration of activating doses of adrenaline, acute phase of immobilization stress, initial period after allogenic transplantation, and acute phase of myocardial infarction) or with prevalence of the reciprocal mediator acetylcholine (late period after transplantation, chronic ulcer disease of stomach and duodenum). Adrenaline prevalence leads to increase of succinate-dependent ATP synthesis and Ca2+ accumulation, which is due to known activation of succinate oxidation. However, together with activation, inhibition of these processes was revealed. The inhibition phase prevails under chronic pathology. Therefore, reciprocal regulation of succinate oxidation and succinate-dependent Ca2+ transport in mitochondria occurs in the body in the course of adaptation. The reciprocal regulation of mitochondrial processes is considered as a mechanism of reciprocal regulation of physiological functions.

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin

Objective: This study was designed to compare ischemic preconditioning with opening of mitochondrial adenosine triphosphate-sensitive potassium channels and Na(+)/H(+) exchange inhibition in an isolated heart model of cold storage, simulating the situation of cardiac allografts., Methods: Sixty-seven isolated isovolumic buffer-perfused rat hearts were arrested with and stored in Celsior solution (Imtix-Sangstat) at 4 degrees C for 4 hours before a 2-hour reperfusion. Group I hearts served as controls and were arrested with and stored in Celsior solution. In group II, hearts were preconditioned by two 5-minute episodes of global ischemia, each separated by 5 minutes of reperfusion before arrest with Celsior solution. Group III hearts were arrested with and stored in Celsior solution supplemented with 100 micromol/L of the mitochondrial adenosine triphosphate-sensitive potassium channel opener diazoxide. In group IV, hearts received an infusion of diazoxide (30 micromol/L) during the first 15 minutes of reperfusion. Group V hearts underwent a protocol combining both interventions used in groups III and IV. In group VI, hearts were arrested with and stored in Celsior solution supplemented with 1 micromol/L of the Na(+)/H(+) exchange inhibitor cariporide. Group VII hearts received an infusion of cariporide (1 micromol/L) during the first 15 minutes of reperfusion. In group VIII, hearts underwent a protocol combining both interventions used in groups VI and VII. Group IX hearts were ischemically preconditioned as in group II, and sustained Na(+)/H(+) exchange inhibition during both storage and early reperfusion was used as in group VIII., Results: On the basis of comparisons of postischemic left ventricular contractility and diastolic function, coronary flow, total creatine kinase leakage, and myocardial water content, values indicative of improved protection were obtained by combining ischemic preconditioning with Na(+)/H(+) exchange inhibition by cariporide given during storage and initial reperfusion. The endothelium-dependent vasodilatory postischemic responses to 5-hydroxytryptamine or acetylcholine and endothelium-independent responses to papaverine were not affected by these interventions., Conclusions: These data suggest that cardioprotection conferred by the Na(+)/H(+) exchange inhibitor cariporide is additive to that of ischemic preconditioning and might effectively contribute to improve donor heart preservation during cardiac transplantation.

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras., (Copyright 2000 Academic Press.)

1. The ATP action on spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) was investigated in rat substantia gelatinosa (SG) neurons mechanically dissociated from the 2nd layer of the dorsal horn in which their presynaptic glycinergic nerve terminals remained intact. 2. ATP reversibly facilitated the frequency of the mIPSCs in a concentration-dependent manner without affecting their amplitude distribution. The ATP agonist, 2-methylthioATP (2MeSATP), mimicked the ATP action, while another ATP receptor agonist, alphabeta-methylene-ATP (alpha,beta-meATP), had no effect on mIPSCs. 3. The ATP receptor antagonists, suramin (1 x 10-6 M) and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (1 x 10-5 M), completely blocked the facilitatory effect of ATP on glycine release (102.0 +/- 11.2 % and 99.3 +/- 16.2 %, n = 6, respectively) without altering the current amplitude distributions. 4. N-Ethylmaleimide (NEM), a sulphydryl alkylating agent, suppressed the inhibitory effect of adenosine on mIPSC frequency (111.2 +/- 13. 3 %, n = 4) without altering the current amplitude distribution. However, ATP still facilitated the mIPSC frequency (693.3 +/- 245.2 %, n = 4) even in the presence of NEM. 5. The facilitatory effect of ATP (1 x 10-5 M) on mIPSC frequency was not affected by adding 1 x 10-4 M Cd2+ to normal external solution but was eliminated in a Ca2+-free external solution. 6. These results suggest that ATP enhances glycine release from nerve terminals, presumably resulting in the inhibition of SG neurons which conduct nociceptive signals to the CNS. This presynaptic P2X-type ATP receptor may function to prevent excess excitability in SG neurons, thus preventing an excessive pain signal and/or SG cell death.

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 microM), ATP was approximately 10-fold less potent (IC50 = 9.1-21.2 microM) than UTP (IC50 = 0.7-2.9 microM) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.

Background: The aim of the study was to evaluate the efficacy of ATP-MgCl2 on myocardial insufficiency associated with hypovolemic shock in dogs. We designed the study as a controlled randomized study., Methods: Six mixed-breed dogs weighing 22 +/- 3 kg were included in the control group and 20 +/- 3 kg in the ATP-MgCl2 group. After the animals were anesthetized 40 ml/kg of blood was withdrawn in 15 minutes. Animals were observed for 45 minutes after removal of blood. Six animals received 45 ml/kg of lactated Ringer's solution and the other animals were treated with 45 ml/kg of lactated Ringer's solution and ATP-MgCl2. All measurements were made before removal of blood, 45 min after exsanguination and at 1 hour intervals for 3 hours. The following parameters were measured; systemic and pulmonary arterial pressures, pulmonary capillary wedge pressure, central venous pressure, cardiac output, rectal temperature, arterial pH, PCO2 and PO2 and mixed venous hemoglobin oxygen saturation. In addition blood samples were collected for the analysis of lactate and tumor necrosis factor (TNF) concentrations., Results: After hemorrhage, cardiac index (CI) decreased significantly from 122 +/- 9 to 52 +/- 9 ml/kg/min in the control group (p < 0.0001) and from 124 +/- 11 ml/kg/min to 50 +/- 6 ml/kg/min in the ATP-MgCl2 group, respectively (p < 0.0001). After volume replacement, Cl was 93 +/- 6 ml/kg/min in the control group and 111 +/- 4 ml/kg/min in the ATP-MgCl2 group 3 hours after the onset of reinfusion, respectively (p < 0.05). TNF was 36 +/- 5 pg/ml in the control group and 21 +/- 3 pg/ml in the ATP-MgCl2 group (p < 0.05). Three hours after the onset of hemorrhagic shock, oxygen consumption and delivery were 126 +/- 14 and 206 +/- 19 ml/min in the control group and 198 +/- 16 and 305 +/- 27 ml/min in the ATP-MgCl2 group, respectively. At the same time point the oxygen extraction ratio was 0.49 +/- 0.04 in the control group and 0.61 +/- 0.03 in the ATP-MgCl2 group (p < 0.01)., Conclusions: Hemorrhagic shock causes TNF release which may cause multiple organ failure. Organ dysfunction still persists even after the appropriate treatment. ATP-MgCl2 attenuates the release of TNF which may improve the adverse effects of hemorrhagic shock.

P1,P4-Diadenosine 5'-tetraphosphate (Ap4A) acts as an extracellular modulator through its interaction with purinoceptors. Our laboratory has demonstrated the presence of an Ap4A receptor in cardiac tissue [1,2]. Due to the rapid hydrolysis of ATP by cardiac membranes the relationship of ATP and Ap4A binding to purinoceptors on cardiac membranes has not been characterized. In this communication we used two approaches to determine the relationship of ATP to the Ap4A receptor. Radioligand binding carried out with [alpha-32P]Ap4A and adenosine 5'-O-¿3-thiotriphosphate¿ ([gamma-35S]ATPgammaS) demonstrates the presence of a single high affinity binding site for Ap4A and the presence of two binding sites for ATPgammaS. The second approach utilized immunoaffinity purified Ap4A receptor that was shown to be free of ATPase and Ap4Aase activities. Non-radiolabeled Ap4A and ATPgammaS effectively inhibited photocrosslinking of [alpha-32P]8-N3Ap4A to the receptor polypeptide while ATP was a much less effective inhibitor. Furthermore, on plasma membranes [alpha-32P]8-N3Ap4A photocrosslinked to only a 50 kDa polypeptide. These data are consistent with Ap4A interacting with a homogeneous population of receptors on cardiac plasma membranes but with ATP having a low affinity for the receptor.

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.