Depletion Of Extracellular ATP Research Articles

Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.

ATP is actively maintained at high concentrations in cancerous tissues, where it promotes a malignant phenotype through P2 receptors. In this study, we first evaluated the effect of extracellular ATP depletion with apyrase in SKOV-3, a cell line derived from metastatic ovarian carcinoma. We observed a decrease in cell migration and an increase in transepithelial electrical resistance and cell markers, suggesting a role in maintaining a mesenchymal phenotype. To identify the P2 receptor that mediated the effects of ATP, we compared the transcript levels of some P2 receptors and found that P2RX7 is three-fold higher in SKOV-3 cells than in a healthy cell line, namely HOSE6-3 (from human ovarian surface epithelium). Through bioinformatic analysis, we identified a higher expression of the P2RX7 transcript in metastatic tissues than in primary tumors; thus, P2X7 seems to be a promising effector for the malignant phenotype. Subsequently, we demonstrated the presence and functionality of the P2X7 receptor in SKOV-3 cells and showed through pharmacological approaches that its activity promotes cell migration and contributes to maintaining a mesenchymal phenotype. P2X7 activation using BzATP increased cell migration and abolished E-cadherin expression. On the other hand, a series of P2X7 receptor antagonists (A438079, BBG and OxATP) decreased cell migration. We used a CRISPR-based knock-out system directed to P2RX7. According to the results of our wound-healing assay, SKOV3-P2X7KO cells lacked receptor-mediated calcium mobilization and decreased migration. Altogether, these data let us propose that P2X7 receptor is a regulator for cancer cell migration and thus a potential drug target.

NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1β (interleukin-1β) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1β production to induce adaptive hypertrophy in the stressed heart. C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1β antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1β and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. Genetic disruption of NLRP3 resulted in significant loss of IL-1β production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1β production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1β-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic β-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1β production, and adaptive cardiac hypertrophy during pressure overload. Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

Involvement of ceramide biosynthesis in increased extracellular vesicle release in Pkd1 knock out cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.

Mice Lacking the Purinergic Receptor P2X5 Exhibit Defective Inflammasome Activation and Early Susceptibility to Listeria monocytogenes.

P2X5 is a member of the P2X purinergic receptor family of ligand-gated cation channels and has recently been shown to regulate inflammatory bone loss. In this study, we report that P2X5 is a protective immune regulator during Listeria monocytogenes infection, as P2X5-deficient mice exhibit increased bacterial loads in the spleen and liver, increased tissue damage, and early (within 3-6 d) susceptibility to systemic L. monocytogenes infection. Whereas P2X5-deficient mice experience normal monocyte recruitment in response to L. monocytogenes, P2X5-deficient bone marrow-derived macrophages (BMMs) exhibit defective cytosolic killing of L. monocytogenes We further showed that P2X5 is required for L. monocytogenes-induced inflammasome activation and IL-1β production and that defective L. monocytogenes killing in P2X5-deficient BMMs is substantially rescued by exogenous IL-1β or IL-18. Finally, in vitro BMM killing and in vivo L. monocytogenes infection experiments employing either P2X7 deficiency or extracellular ATP depletion suggest that P2X5-dependent anti-L. monocytogenes immunity is independent of the ATP-P2X7 inflammasome activation pathway. Together, our findings elucidate a novel and specific role for P2X5 as a critical mediator of protective immunity.

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP

Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated Tcell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.

Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.

Depletion of extracellular ATP affects the photosystem II photochemistry and the role of salicylic acid in this process

In the present work, treatment with AMP-PCP (β,γ-methyleneadenosine-5'-triphosphate - a competitive inhibitor of the extracellular ATP pool) or apyrase (an ATP-degrading enzyme) decreased the PSII operating efficiency (ΦPSII), photochemical quenching (qp), and maximum quantum efficiency of PSII photochemistry (Fv/Fm) of Arabidopsis leaves illuminated with high light intensity. Mutation of extracellular ATP receptor, DORN1, also caused decreases of ΦPSII, qp, and Fv/Fm when the leaves were exposed to high light. Either the treatment with AMP-PCP and apyrase or the mutation in DORN1 increased the salicylic acid content in the illuminated leaves. Salicylic acid deficiency in NahG transgenic mutant made the PSII photochemistry more sensitive to AMP-PCP or apyrase. These results indicate that extracellular ATP depletion can affect the PSII photochemistry, whereas salicylic acid can relieve this effect.

Role of ATP during the initiation of microvascularization: acceleration of an autocrine sensing mechanism facilitating chemotaxis by inorganic polyphosphate.

The in vitro tube formation assay with human umbilical vein endothelial cells (HUVEC) was applied to identify the extra- and intracellular sources of metabolic energy/ATP required for cell migration during the initial stage of microvascularization. Extracellularly, the physiological energy-rich polymer, inorganic polyphosphate (polyP), applied as biomimetic amorphous calcium polyP microparticles (Ca-polyP-MP), is functioning as a substrate for ATP generation most likely via the combined action of the alkaline phosphatase (ALP) and the adenylate kinase (AK). The linear Ca-polyP-MP with a size of 40 phosphate units, close to the polyP in the acidocalcisomes in the blood platelets, were found to increase endothelial cell tube formation, as well as the intracellular ATP levels. Depletion of extracellular ATP with apyrase suppressed tube formation during the initial incubation period. Inhibition experiments revealed that inhibitors (levamisole and Ap5A) of the enzymes involved in extracellular ATP generation strongly reduce the Ca-polyP-MP-induced tube formation. The stimulatory effect of Ca-polyP-MP was also diminished by the glycolysis inhibitor oxamate and trifluoperazine which blocks endocytosis, as well as by MRS2211, an antagonist of the P2Y13 receptor. Oligomycin, an inhibitor of the mitochondrial F0F1-ATP synthase, displayed no effect at lower concentrations on tube formation. Electron microscopic data revealed that after cellular uptake, the Ca-polyP-MP accumulate close to the cell membrane. We conclude that in HUVEC exposed to polyP, ATP is formed extracellularly via the coupled ALP-AK reaction, and intracellularly during glycolysis. The results suggest an autocrine signaling pathway of ATP with polyP as an extracellular store of metabolic energy for endothelial cell migration during the initial vascularization process.

Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica)

At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

ClC‐3 is a main component of background chloride channels activated under isotonic conditions by autocrine ATP in nasopharyngeal carcinoma cells

In this study, the activation mechanisms of the background chloride current and the role of the current in maintaining of basal cell volume were investigated in human nasopharyngeal carcinoma CNE-2Z cells. Under isotonic conditions, a background chloride current was recorded by the patch clamp technique. The current presented the properties similar to those of the volume-activated chloride current in the same cell line and was inhibited by chloride channel blockers or by cell shrinkage induced by hypertonic challenges. Extracellular applications of reactive blue 2, a purinergic receptor antagonist, suppressed the background chloride current in a concentration-dependent manner under isotonic conditions. Depletion of extracellular ATP with apyrase or inhibition of ATP release from cells by gadolinium chloride decreased the background current. Extracellular applications of micromolar concentrations of ATP activated a chloride current which was inhibited by chloride channel blockers and hypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride. Transfection of CNE-2Z cells with ClC-3 siRNA knocked down expression of ClC-3 proteins, attenuated the background chloride current and prevented activation of the ATP-induced current. Furthermore, knockdown of ClC-3 expression or exposures of cells to ATP (10 mM), the chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, or reactive blue 2 increased cell volume under isotonic conditions. The results suggest that ClC-3 protein may be a main component of background chloride channels which can be activated under isotonic conditions by autocrine/paracrine ATP through purinergic receptor pathways; the background current is involved in maintenance of basal cell volume.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity.

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Proteomic Analysis of Extracellular ATP-Regulated Proteins Identifies ATP Synthase β-Subunit as a Novel Plant Cell Death Regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.

Extracellular ATP Functions as an Endogenous External Metabolite Regulating Plant Cell Viability

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with beta,gamma-methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

Purinergic-independent Calcium Signaling Mediates Recovery from Hepatocellular Swelling: IMPLICATIONS FOR VOLUME REGULATION

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.