Exogenous ATP Research Articles

Role of apyrase-mediated eATP signal in chilling injury of postharvest banana fruit during storage

Apyrase (APY) might regulate the homeostasis of extracellular ATP (eATP) / intracellular ATP (iATP) and mediate plant growth and defense response, but little information on the roles of APY genes in eATP-mediated chilling response of banana fruit during storage has been available. This study identified a total of 10 full-length banana APY genes, analyzed cis-elements in promoters and potential miRNA target sites and found that these APYs had highly conserved functions and might regulate energy metabolism during banana fruit development, ripening and senescence. Exogenous ATP was used to investigate the roles of APY genes in chilling injury (CI) of banana fruit stored for up to 6 d at 6 °C. Application of 1 mM ATP solution effectively reduced CI symptoms, inhibited malondialdehyde (MDA) accumulation, improved antioxidant capacity, and relatively maintained the homeostasis of iATP / eATP of banana fruit during cold storage. Compared with non-ATP-treated fruit, transcriptional levels of MaAPYs were up-regulated whereas those of nucleoside transporter genes and purine permease genes were down-regulated in ATP-treated banana fruit during cold storage, suggesting that the ATP pretreatment could maintain the relative homeostasis of iATP / eATP. The results provided the new insights into the roles of apyrase-mediated eATP signal in regulating the cold tolerance of banana fruit during storage at low temperature.

Extracellular ATP is involved in regulating Arabidopsis seed germination.

Extracellular ATP level induced a transient increase during germination of Arabidopsis seeds, and extracellular ATP could negatively regulate the seed germination by its receptor, DORN1. Extracellular ATP (exATP) acts as a signal molecule for regulating growth, development, and responses of plants to external environments. In this study, we investigated the possible involvement of exATP in regulating the seed germination of Arabidopsis thaliana. Treatments of Arabidopsis seeds with exogenous ATP delayed seed germination, suggesting that exATP could be a repressor for seed germination. During the germination of Arabidopsis seeds, the exATP level of the seeds presented a transient increase. When exogenous application of the glucose-hexokinase system effectively decreased the exATP level of the Arabidopsis seeds during germination, the percentage of germination was significantly enhanced, while the products of ATP hydrolysis had no effects on the germination. Further studies showed that the seeds of dorn 1-3 mutant plants (mutation in exATP receptor) showed a higher germination percentage, compared to the seeds of wide type (WT) plants. In addition, the dorn 1-3 mutant seeds were less sensitive to the delay-effect of exogenous ATP on seed germination than the WT seeds. The dorn 1-3 mutant seeds presented a higher GA (gibberellin) content, lower ABA (abscisic acid) content, and lower ratio of ABA/GA contents before the imbibition, compared to the WT seeds. The regulation of seed germination by exATP was dependent on the external temperature. These data suggest that exATP is involved in regulating Arabidopsis seed germination.

A perylene diimide-based nanoring architecture for exogenous and endogenous ATP detection: biochemical assay for monitoring phosphorylation of glucose.

Positively charged amphiphiles hold great significance in supramolecular chemistry due to their good solubility, and physiochemical and molecular recognition properties. Herein, we report the synthesis, characterization and molecular recognition properties of the dicationic amphiphile based on perylene diimide-tyrosine alkyl amide amine (PDI 3). PDI 3 showed the formation of a nanoring architecture in the self-assembled aggregated state (90% H2O-DMSO mixture) as observed by SEM and TEM studies. The diameter of the nanoring is around 30-50 nm with a height varying from 1 to 2 nm. The self-assembled aggregates of PDI 3 are very sensitive towards nucleoside triphosphates. Upon addition of ATP, PDI 3 showed a decrease in the absorbance and emission intensity at 535 and 580 nm (due to the monomer state), respectively. The lowest detection limit for ATP is 10.8 nM (UV) and 3.06 nM (FI). Upon interaction of ATP with PDI 3, the nanoring morphology transformed into a spherical structure. These changes could be attributed to the formation of ionic self-assembled aggregates between dicationic PDI 3 and negatively charged ATP via electrostatic and H-bonding interactions. The complexation mechanism of PDI 3 and ATP was confirmed by optical, NMR, Job's plot, DLS, SEM and AFM studies. PDI 3 displays low cytotoxicity toward MG-63 cells and can be successfully used for the detection of exogenous and endogenous ATP. The resulting PDI 3 + ATP complex is successfully used as a 'turn-on' biochemical assay for monitoring phosphorylation of glucose.

Histamine enhances ATP-induced itching and responsiveness to ATP in keratinocytes

Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca2+]i) in stimulated cells. This action propagates a Ca2+ wave to neighboring keratinocytes via ATP/P2Y2 receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X3 receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X3 receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca2+]i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.

Mitochondrial alternative oxidase pathway acts as an electron sink during photosynthetic induction in Rumex K-1 leaves

Chloroplast PSII photochemical efficiency is upregulated more rapidly than CO2 assimilation during photosynthesis induction, suggesting the existence of other electron sinks than that of CO2 assimilation. We hypothesized that the mitochondrial alternative oxidase (AOX) pathway could be such a sink. Inhibition of the AOX restricted light activation of the malate-oxaloacetate shuttle and caused an excessive reduction of PSI acceptor side and substantial accumulation of QA-, hindering the photosynthetic linear electron transport rate (ETR) and leading to an imbalance between light energy absorption and exploitation during photosynthetic induction. ETR limitation also restricted the formation of thylakoid pH gradient, evidenced by a decreased de-epoxidation of the xanthophyll cycle, thus preventing nonphotochemical quenching. Delayed CO2 assimilation due to thylakoid pH gradient restriction was partially reversed by exogenous ATP application. The AOX pathway acts as a photosynthetic electron sink, protecting the photosynthetic apparatus against photoinhibition and accelerating the induction of CO2 assimilation during photosynthetic induction in Rumex K-1 leaves.

L-type Ca2+ Channels at Low External Calcium Differentially Regulate Neurotransmitter Release in Proximal-Distal Compartments of the Frog Neuromuscular Junction.

L-type Ca2+ channels (LTCCs) are key elements in electromechanical coupling in striated muscles and formation of neuromuscular junctions (NMJs). However, the significance of LTCCs in regulation of neurotransmitter release is still far from understanding. Here, we found that LTCCs can increase evoked neurotransmitter release (especially asynchronous component) and spontaneous exocytosis in two functionally different compartment of the frog NMJ, namely distal and proximal parts. The effects of LTCC blockage on evoked and spontaneous release as well as timing of exocytotic events were prevented by inhibition of either protein kinase C (PKC) or P2Y receptors (P2Y-Rs). Hence, endogenous signaling via P2Y-R/PKC axis can sustain LTCC activity. Application of ATP, a co-neurotransmitter able to activate P2Y-Rs, suppressed both evoked and spontaneous exocytosis in distal and proximal parts. Surprisingly, inhibition of LTCCs (but not PKC) decreased the negative action of exogenous ATP on evoked (only in distal part) and spontaneous exocytosis. Lipid raft disruption suppressed (1) action of LTCC antagonist on neurotransmitter release selectively in distal region and (2) contribution of LTCCs in depressant effect of ATP on evoked and spontaneous release. Thus, LTCCs can enhance and desynchronize neurotransmitter release at basal conditions (without ATP addition), but contribute to ATP-mediated decrease in the exocytosis. The former action of LTCCs relies on P2Y-R/PKC axis, whereas the latter is triggered by exogenous ATP and PKC-independent. Furthermore, relevance of lipid rafts for LTCC function as well as LTCCs for ATP effects is different in distal and proximal part of the NMJ.

Endothelial pannexin 1-TRPV4 channel signaling lowers pulmonary arterial pressure in mice.

Pannexin 1 (Panx1), an ATP-efflux pathway, has been linked with inflammation in pulmonary capillaries. However, the physiological roles of endothelial Panx1 in the pulmonary vasculature are unknown. Endothelial transient receptor potential vanilloid 4 (TRPV4) channels lower pulmonary artery (PA) contractility and exogenous ATP activates endothelial TRPV4 channels. We hypothesized that endothelial Panx1-ATP-TRPV4 channel signaling promotes vasodilation and lowers pulmonary arterial pressure (PAP). Endothelial, but not smooth muscle, knockout of Panx1 increased PA contractility and raised PAP in mice. Flow/shear stress increased ATP efflux through endothelial Panx1 in PAs. Panx1-effluxed extracellular ATP signaled through purinergic P2Y2 receptor (P2Y2R) to activate protein kinase Cα (PKCα), which in turn activated endothelial TRPV4 channels. Finally, caveolin-1 provided a signaling scaffold for endothelial Panx1, P2Y2R, PKCα, and TRPV4 channels in PAs, promoting their spatial proximity and enabling signaling interactions. These results indicate that endothelial Panx1-P2Y2R-TRPV4 channel signaling, facilitated by caveolin-1, reduces PA contractility and lowers PAP in mice.

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells.

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

The lamprey respiratory network: Some evolutionary aspects

Breathing is a complex behaviour that involves rhythm generating networks. In this review, we examine the main characteristics of respiratory rhythm generation in vertebrates and, in particular, we describe the main results of our studies on the role of neural mechanisms involved in the neuromodulation of the lamprey respiration. The lamprey respiratory rhythm generator is located in the paratrigeminal respiratory group (pTRG) and shows similarities with the mammalian preBötzinger complex. In fact, within the pTRG a major role is played by glutamate, but also GABA and glycine display important contributions. In addition, neuromodulatory influences are exerted by opioids, substance P, acetylcholine and serotonin. Both structures respond to exogenous ATP with a biphasic response and astrocytes there located strongly contribute to the modulation of the respiratory pattern. The results emphasize that some important characteristics of the respiratory rhythm generating network are, to a great extent, maintained throughout evolution.

Autocrine regulation of wound healing by ATP release and P2Y2 receptor activation

AimsApplication of exogenous nucleotides can modulate wound healing via the activation of purinergic receptors. However, evidence for the release of endogenous nucleotides and the subsequent activation of purinergic receptors in this process has not been well defined. Therefore, the current study aimed to investigate wound-mediated nucleotide release and autocrine purinergic signalling during HaCaT keratinocyte wound closure following scratch injury. Main methodsAn in vitro scratch wound apparatus was employed to study wound healing over 24-h in the presence of modulators of ATP release, P2 receptors and pathways downstream of P2 receptor activation. Key findingsAdenosine 5′-triphosphate (ATP) was released from scratched cells. The ectonucleotidase apyrase and pharmacological inhibition of the nucleotide release hemichannel, pannexin-1, decreased wound closure over time. The non-selective P2Y receptor antagonist suramin and the selective P2Y2 receptor antagonist AR-C118925XX, but not other P2 antagonists, decreased wound closure. AR-C118925XX decreased wound closure in a concentration-dependent fashion. However, exogenous P2Y2 receptor agonists, ATP or uridine 5′-triphosphate, did not enhance wound closure. PCR and immunoblotting confirmed P2Y2 receptor expression in HaCaT cells. U73122, a phospholipase C antagonist, and 2-aminoethoxydiphenylborate, an inositol 1,4,5-trisphosphate receptor-sensitive Ca2+-release channel antagonist, decreased wound closure consistent with P2Y2 receptor activation. Absence of extracellular or intracellular Ca2+ or inhibition of intracellular Ca2+-release also impaired wound closure. SignificanceThese data describe a novel autocrine signalling mechanism in which wound-mediated release of endogenous ATP in response to mechanical scratching of HaCaT cells activates P2Y2 receptors to facilitate wound closure.

The signaling role of extracellular ATP in co-culture of Shiraia sp. S9 and Pseudomonas fulva SB1 for enhancing hypocrellin A production

BackgroundAdenosine 5′-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture.ResultsThe co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10–300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture.ConclusionsOur results indicate that eATP release is an early event during the intimate bacterial–fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal–bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites.Graphic abstract

Isolation of Arabidopsis extracellular ATP binding proteins by affinity proteomics and identification of PHOSPHOLIPASEC-LIKE1 as an extracellular protein essential for fumonisinB1 toxicity.

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE52, SUBTILASE-LIKE SERINE PROTEASE1.7 and PHOSPHOLIPASEC-LIKE1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisinB1, a cell death-activating mycotoxin. The expression of PEROXIDASE52 and PHOSPHOLIPASEC-LIKE1 was altered in fumonisinB1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisinB1 suppressed PHOSPHOLIPASEC-LIKE1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASEC-LIKE1 were resistant to fumonisinB1-induced death. The activation of PHOSPHOLIPASEC-LIKE1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASEC-LIKE1 serves as a convergence point for fumonisinB1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisinB1.

Abacavir Increases Purinergic P2X7 Receptor Activation by ATP: Does a Pro-inflammatory Synergism Underlie Its Cardiovascular Toxicity?

The cardiovascular toxicity of Abacavir is related to its purinergic structure. Purinergic P2X7-receptors (P2X7R), characterized by activation by high concentrations of ATP and with high plasticity, seem implicated. We appraise the nature of the interplay between Abacavir and P2X7R in generating vascular inflammation. The effects of Abacavir on leukocyte-endothelium interactions were compared with those of its metabolite carbovir triphosphate (CBV-TP) or ATP in the presence of apyrase (ATP-ase) or A804598 (P2X7R-antagonist). CBV-TP and ATP levels were evaluated by HPLC, while binding of Abacavir, CBV-TP and ATP to P2X7R was assessed by radioligand and docking studies. Hypersensitivity studies explored a potential allosteric action of Abacavir. Clinical concentrations of Abacavir (20 µmol/L) induced leukocyte-endothelial cell interactions by specifically activating P2X7R, but the drug did not show affinity for the P2X7R ATP-binding site (site 1). CBV-TP levels were undetectable in Abacavir-treated cells, while those of ATP were unaltered. The effects of Abacavir were Apyrase-dependent, implying dependence on endogenous ATP. Exogenous ATP induced a profile of proinflammatory actions similar to Abacavir, but was not entirely P2X7R-dependent. Docking calculations suggested ATP-binding to sites 1 and 2, and Abacavir-binding only to allosteric site 2. A combination of concentrations of Abacavir (1 µmol/L) and ATP (0.1 µmol/L) that had no effect when administered separately induced leukocyte-endothelium interactions mediated by P2X7R and involving Connexin43 channels. Therefore, Abacavir acts as a positive allosteric modulator of P2X7R, turning low concentrations of endogenous ATP themselves incapable of stimulating P2X7R into a functional proinflammatory agonist of the receptor.

Micromechanical properties and functional activity of granulocytes when simulating exogenous loading with ATP in vitro

The micromechanical properties of leukocytes make a certain contribution to the blood flow velocity in the microcirculatory bed, while the micromechanical properties themselves change under the influence of a complex network of purinergic signals.The aim of the work was to study the micromechanical properties and functional activity of granulocytes in normal conditions and in patients with acute lymphoblastic leukemia when simulating exogenous loading with ATP in vitro.Materials and methods. Leukocytes were isolated from the blood of patients with acute lymphoblastic leukemia and healthy people. Each sample was divided into a test sample and a control sample. In the test samples, the loading with ATP in vitro was simulated. Leukocytes of the control samples were incubated in the culture medium without the addition of ATP. Young’s modulus and adhesion force were measured using an atomic force microscope in the force spectroscopy mode. The cell surface potential was measured in an atomic force microscope in the Kelvin mode. To assess the functional activity of granulocytes, hypoosmotic tests in vitro and determination of migration activity were used.Results. In tests with exogenous ATP, both in samples from healthy people and from patients with acute lymphoblastic leukemia, a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties of leukocytes and migration activity were found. At the same time, the responses of granulocytes to the osmotic loading were different: for example, in the group of healthy people, the loading with ATP caused cell contraction and a decrease in the use of the membrane reserve by the cell in a hypotonic environment, and in patients with acute lymphoblastic leukemia, it caused an increase in the volume and more intensive use of the membrane reserve in volume regulation.Conclusion. The revealed effects indicate the leading role of the ATP molecule in the signal transduction mechanisms between blood cells in the microvasculature. The increase in the adhesive properties of the cell surface of granulocytes revealed in the study, in parallel with the increase in their migration activity under the influence of the ATP molecule, can contribute to the development of inflammation in the vessel wall.

Astrocyte-mediated purinergic signaling is upregulated in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism spectrum disorders. With increasing investigation into the molecular mechanisms underlying FXS, there is growing evidence that perturbations in glial signaling are widely associated with neurological pathology. Purinergic signaling, which utilizes nucleoside triphosphates as signaling molecules, provides one of the most ubiquitous signaling systems for glial-neuronal and glial-glial crosstalk. Here, we sought to identify whether purinergic signaling is dysregulated within the FXS mouse cortex, and whether this dysregulation contributes to aberrant intercellular communication. In primary astrocyte cultures derived from the Fmr1 knockout (KO) mouse model of FXS, we found that application of exogenous ATP and UTP evoked elevated intracellular calcium responses compared to wildtype levels. Accordingly, purinergic P2Y2 and P2Y6 receptor expression was increased in Fmr1 KO astrocytes both in vitro and in acutely dissociated tissue, while P2Y antagonism via suramin prevented intracellular calcium elevations, suggesting a role for these receptors in aberrant FXS astrocyte activation. To investigate the impact of elevated purinergic signaling on astrocyte-mediated synaptogenesis, we quantified synaptogenic protein TSP-1, known to be regulated by P2Y activation. TSP-1 secretion and expression were both heightened in Fmr1 KO vs wildtype astrocytes following UTP application, while naïve TSP-1 cortical expression was also transiently elevated in vivo, indicating increased potential for excitatory TSP-1-mediated synaptogenesis in the FXS cortex. Together, our results demonstrate novel and significant purinergic signaling elevations in Fmr1 KO astrocytes, which may serve as a potential therapeutic target to mitigate the signaling aberrations observed in FXS.

Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

Exogenous ATP modulates PGE2 release in macrophages through sustained phosphorylation of CDK9 and p38 MAPK.

An important mediator of inflammation is prostaglandin E2 (PGE2 ), whose levels are determined by the activity of the enzyme cyclooxygenase (COX). Of the two isoforms of the enzyme, COX-2 has been shown to be induced in macrophages during inflammation. Although general COX inhibitors, belonging to the class of nonsteroidal anti-inflammatory drugs, or specific COX-2 inhibitors, called coxibs, are useful in the control of acute inflammation, adverse reactions were seen when used chronically in the treatment of rheumatoid arthritis or neurodegenerative diseases. Extracellular ATP (eATP) has been reported as a damage-associated molecular pattern signal. In this report, we show that eATP synergistically increases the levels of COX-2 enzyme and PGE2 in LPS-activated RAW264.7 macrophages and human monocytes. Activation of macrophages also occurred when cultured in media obtained from dying neurons that contained higher levels of ATP. We show that eATP increases the levels of COX-2 protein, which is sustained up to 36 h poststimulation. This is in turn due to sustained levels of phosphorylated, or activated, cyclin-dependent kinase 9 and p38 MAPK in ATP-treated cells compared to LPS-stimulated cells. The eATP-dependent increase in COX-2/PGE2 levels in LPS-activated RAW264.7 cells could be abolished using antagonists for purinergic P2X7-and P2Y6 receptors. Similarly, the increase in COX-2/PGE2 levels in the peritoneum of LPS-treated mice could be significantly abolished in mice that were preinjected with the nonspecific P2 receptor antagonist, suramin. P2 receptor antagonists, therefore, should be explored in our search for an ideal anti-inflammatory candidate.

БИОФИЗИЧЕСКИЕ СВОЙСТВА КЛЕТОЧНОЙ ПОВЕРХНОСТИ И ФУНКЦИОНАЛЬНАЯ АКТИВНОСТЬ ГРАНУЛОЦИТОВ БОЛЬНЫХ ОСТРЫМ ЛИМФОБЛАСТНЫМ ЛЕЙКОЗОМ ПРИ МОДЕЛИРОВАНИИ НАГРУЗКИ С АТФ IN VITRO

The current study is devoted to the investigation of the biophysical properties of the cell surface and the functional activity of granulocytes in patients with acute lymphoblastic leukemia when simulating the ATP load in vitro. The experiment was performed on the peripheral blood of patients with acute lymphoblastic leukemia (ALL) who underwent a standard course of chemotherapy. In experimental tests, exogenous loading with ATP was simulated in vitro by adding 100.0 μM adenosine-5-triphosphate disodium salt trihydrate to the granulocyte suspension. Incubation with the drug was carried out for 15 min at 370 C. As a control, a suspension of granulocytes in RPMI 1640 medium from the same patient, but without the addition of the drug, was used, this was incubated for 15 min at a temperature of 370 C. After the incubation time, the biophysical properties (rigidity, charge of the cell surface, the strength of intercellular adhesion) of granulocytes in experimental and control samples were studied using atomic force microscopy, and the migration activity of cells was also assessed in a direct capillary test under agarose. A model with exogenous ATP in ALL patients showed a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties and migration activity of granulocytes. The revealed effects point to the key role of the ATP molecule in the mechanisms of intercellular signaling in the microvasculature.

Interaction of amyloid beta with humanin and acetylcholinesterase is modulated by ATP

Humanin (HN) is known to bind amyloid beta (Aβ)-inducing cytoprotective effects, while binding of acetylcholinesterase (AChE) to Aβ increases its aggregation and cytotoxicity. Previously, we showed that binding of HN to Aβ blocks aggregation induced by AChE and that HN decreases but does not abolish Aβ-AChE interactions in A549 cell media. Here, we set out to shed light on factors that modulate the interactions of Aβ with HN and AChE. We found that binding of either HN or AChE to Aβ is not affected by heparan sulfate, while ATP, thought to reduce misfolding of Aβ, weakened interactions between AChE and Aβ but strengthened those between Aβ and HN. Using media from either A549 or H1299 lung cancer cells, we observed that more HN was bound to Aβ upon addition of ATP, while levels of AChE in a complex with Aβ were decreased by ATP addition to A549 cell media. Exogenous addition of ATP to either A549 or H1299 cell media increased interactions of endogenous HN with Aβ to a comparable extent despite differences in AChE expression in the two cell lines, and this was correlated with decreased binding of exogenously added HN to Aβ. Treatment with exogenous ATP had no effect on cell viability under all conditions examined. Exogenously added ATP did not affect viability of cells treated with AChE-immunodepleted media, and there was no apparent protection against the cytotoxicity resulting from immunodepletion of HN. Moreover, exogenously added ATP had no effect on the relative abundance of oligomer versus total Aβ in either cell line.

Oxidative stress induced by the anti-cancer agents, plumbagin, and atovaquone, inhibits ion transport through Na+/K+-ATPase

Oxidative stress inhibits Na+/K+-ATPase (NKA), the ion channel that maintains membrane potential. Here, we investigate the role of oxidative stress-mediated by plumbagin and atovaquone in the inhibition of NKA activity. We confirm that plumbagin and atovaquone inhibit the proliferation of three human (OVCAR-3, SKOV-3, and TYKNu) and one mouse (ID8) ovarian cancer cell lines. The oxygen radical scavenger, N-acetylcysteine (NAC), attenuates the chemotoxicity of plumbagin and atovaquone. Whole-cell patch clamping demonstrates that plumbagin and atovaquone inhibit outward and the inward current flowing through NKA in SKOV-3 and OVCAR-3. Although both drugs decrease cellular ATP; providing exogenous ATP (5 mM) in the pipet solution used during patch clamping did not recover NKA activity in the plumbagin or atovaquone treated SKOV-3 and OVCAR-3 cells. However, pretreatment of the cells with NAC completely abrogated the NKA inhibitory activity of plumbagin and atovaquone. Exposure of the SKOV-3 cells to either drug significantly decreases the expression of NKA. We conclude that oxidative stress caused by plumbagin and atovaquone degrades NKA, resulting in the inability to maintain ion transport. Therefore, when evaluating compounds that induce oxidative stress, it is important to consider the contribution of NKA inhibition to their cytotoxic effects on tumor cells.