Increased ATP Release Research Articles

Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. The brain-specific Lrp4 conditional knockout mice (Lrp4GFAP-Cre), astrocytic-specific Lrp4 conditional knockout mice (Lrp4GFAP-creER), and their control mice (Lrp4f/f) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A2AR (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4GFAP-Cre and Lrp4GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P2X7R or adenosine-A2AR signaling diminished Lrp4GFAP-creER's protective effect. The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A2AR signaling.

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release

BackgroundConnexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease. MethodsRetinal pigment epithelial cells were exposed to HG, 10ng/mL pro-inflammatory cytokines IL-1β and TNF-α, or a combination of both. Quantitative Cytometric Bead Array analysis was used to measure the release of inflammatory cytokines IL-6, IL-8, MCP-1, and sICAM-1, as well as VEGF and ATP. To determine the role of connexin43 hemichannels in the disease process, changes in cytokine and ATP release were evaluated following treatment with Peptide5, a connexin43 hemichannel blocker. Immunohistochemistry was used to compare NLRP3 inflammasome assembly under control and treatment conditions. ResultsCo-application of HG and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF and ATP, to significantly higher levels compared to cytokines alone. Peptide5 prevented cytokine release and prevented the increase in ATP release following co-application of HG and cytokines. Adding exogenous ATP negated Peptide5-mediated protection against inflammatory cytokine release in injury conditions. ConclusionsOur findings show that connexin43 hemichannels play an important role in the amplification and perpetuation of inflammation by mediating an ATP autocrine feedback loop in the inflammasome/inflammation cycle. General significanceTargeting connexin43 hemichannels offers a potential therapeutic strategy to break the inflammatory cycle in diseases such as DR, but also other chronic inflammatory indications.

A novel technology to model pressure-induced cellular injuries in the brain

BackgroundElevated intracranial pressure (ICP) accompanying a number of neurological emergencies is poorly understood, and lacks a model to determine cellular pathophysiology. This limits our ability to identify cellular and molecular biomarkers associated with the pathological progression from physiologic to pathologic ICP. New methodWe developed an ex vivo model of pressure-induced brain injury, which combines 3D neural cell cultures and a newly developed Pressure Controlled Cell Culture Incubator (PC3I). Human astrocytes and neurons maintained in 3D peptide-conjugated alginate hydrogels were subjected to pressures that mimic both physiologic and pathologic levels of ICP for up to 48h to evaluate the earliest impacts of isolated pressure on cellular viability and quantify early indicators of pressure-induced cellular injury. ResultsCompared to control cell cultures grown under physiologic pressure, sustained pathologic pressure exposure increased the release of intracellular ATP in a cell-specific manner. Eighteen hours of sustained pressure resulted in increased ATP release from neurons but not astrocytes. Comparison with existing methodsCell culture incubators maintain cultures at normal atmospheric pressure. Based on multiple literature searches, we are not aware of any other cell culture incubator systems that modify the pressure at which primary CNS cells are maintained. ConclusionThis model simulates the clinical features of elevated ICP encountered in patients with hydrocephalus, and provides a first estimate of the pathological signaling encountered during the earliest perid of progression in neonatal hydrocephalus. This model should provide a means to better understand the pathological biomarkers associated with the earliest stages of elevated ICP.

Evaluation of mesenchymal stem cell modulation of trigeminal neuronal responses to cold

Tissue engineering protocols, such as regenerative endodontic procedures (REPs), comprise biologically based procedures designed to restore normal physiologic function. For REPs, the goal is reconstitution of the pulp-dentin complex by delivering mesenchymal stem cells (MSCs), including the stem cells of the apical papilla (SCAP) into a root canal system. Many patients regain cold sensitivity after REPs, but the mechanism is not understood. We hypothesized that SCAP modulate nociceptive function through a paracrine mechanism that activates cold-sensitive ion channels in neurons. We established a co-culture system with human SCAP and rat trigeminal (TG) sensory neurons in order to determine the effect of SCAP co-culture on neuronal responses using whole-cell patch-clamp electrophysiology. TG neurons co-cultured with SCAP demonstrated increased TRPA1-mediated (p<0.01) and TRPM8-mediated inward current densities (p<0.01) at 24h in co-culture. Cold stimulation to SCAP significantly increased ATP release (p<0.01), and supernatant collected after cold stimulation to SCAP was able to activate cultured TG neurons. Co-culture with SCAP significantly increased sustained ATP-evoked inward current density (p<0.05). These data suggest that SCAP release trophic factors that act on afferent neurons to enhance cold-sensitive ion channel activity.

AB276. SPR-03 In vitro evaluation of hydrostatic pressure on ATP release and Caspase-1 activation in rat urothelial cells

ObjectiveBladder outlet obstruction (BOO) is projected to affect approximately 1.1 billion men and women by 2018. While BOO results from a multitude of etiologies, they are all characterized by chronically elevated storage and voiding pressures, along with inflammation in the bladder tissue, which can lead to bladder fibrosis and overactive bladder symptoms. Previous research in our lab demonstrated that ATP release by rat primary urothelial cells increased in response to elevated pressure (10–15 cmH2O) in vitro. Thus, we hypothesize that the release of ATP by urothelial cells upon exposure to pathological pressures in BOO initiates a cascade of events that includes the formation of the NLRP3 inflammasome and caspase-1 activation. In the present study, we examined the effects of elevated pressure on ATP-purinergic signaling and caspase-1 activation in rat urothelial cells in vitro.MethodsUsing a custom built pressure system, rat urothelial cell line MYP3 cells (1.2×106 cells/well in F-12 K medium) were exposed to pressure conditions that represent both pathological storage and voiding pressures: 15 cmH2O 60 min, 15, 40 and 75 cmH2O 1 min. Cells that were prepared in a similar manner but maintained under atmospheric pressure served as a control. In addition, MYP3 cells that were exposed to a hypotonic condition (240 mOsm) and cells treated with 1.25 mM ATP served as two positive controls. After exposure to pressure, the supernatant media was collected and the extracellular ATP concentration was measured using a luciferin-luciferase assay kit (Life Technologies). Cells were then lysed and intracellular caspase-1 activity was measured using an established method. The pressure experiments (40 cmH2O 1 min) were repeated in the presence of a P2X7 antagonist [100 µM A-438079 (R&D Systems)] to determine the mechanism for pressure-induced caspase-1 activation.ResultsExposure of MYP3 cells to hydrostatic pressure for 1 min at 15, 40 and 75 cmH2O resulted in a 3-, 3.5- and 4.5-fold increase in extracellular ATP levels, respectively, compared to the 0 cmH2O control. When these cells were exposed to a hypotonic solution, ATP release increased by 6-fold compared to the isotonic control. Exposure of MYP3 cells to pressure also resulted in up to 1.3-fold increases in caspase-1 levels, which were similar to the positive controls, indicating caspase-1 levels are maximized after exposure to pathologic pressure conditions.ConclusionsThe significant increase in ATP release indicates that hydrostatic pressure is a good mechanical stimulus to model BOO in vitro. The acute caspase-1 responses after 1-minute exposure to pressure suggest that high-pressure voiding may be an important trigger of the NLRP3 inflammasome in BOO. The results that demonstrate exposure to pressure and treatment with ATP both result in caspase-1 activation implicate autocrine purinergic signaling as a mediator of NLRP inflammasome formation.Funding Source(s)NIH (R01DK103534, P20GM103444), NSF (1264579)

Effect of SQW on the bladder function of mice lacking TRPV1

BackgroundSuo Quan Wan (SQW) is an effective traditional Chinese prescription on treated lower urinary tract symptoms (LUTS), and has been proved have modulation effect on the expression of transient receptor potential vanilloid 1 (TRPV1) in accordance with the recovery of bladder function of overactive bladder rat. This study further investigated the mechanism of SQW modulated TRPV1 signaling and bladder function using TRPV1 knockout (KO) mice.MethodsStudy was conducted using wild type and TRPV1 KO mice. The KO animals were grouped into KO group and SQW treated group. We applied in vivo cystometrogram recording techniques to analyze voiding control of the urinary bladder, as well as in vitro organ bath to study bladder distension response to various compounds, which subsequently elicited normal smooth muscle excitation. Real-time polymerase chain reaction and western blot analysis were performed to quantify the expression of TRPV1 and P2X3 in the bladder. ATP released from bladder strips was measured using the luciferin–luciferase ATP bioluminescence assay kit.ResultsKO preparation inhibited decrease micturition times, while micturition interval and volume were increased. Results of urodynamic record of the TRPV1−/− mice during NS infusion showed reduced bladder pressure and contraction which exhibited decreased response to α, β-me ATP, KCl, and carbachol and no response to CAP. The ATP released by the TRPV1−/− mice from strips of bladder smooth muscles was significantly reduced, along with no TRPV1 expression and reduced expression level of P2X3 in the bladder. SQW could increase ATP release in some degree, while had no effect on TRPV1 and P2X3 expression. SQW could improve bladder pressure slightly, while make no significantly effects on the force response to α,β-meATP, CAP, carbachol in gradient concentration, and KCl, as well as MBC and voiding activities.ConclusionsTRPV1 plays an important role in urinary bladder mechanosensitivity. The effective SQW is hard to play its proper role on bladder function of mice without TRPV1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1420-6) contains supplementary material, which is available to authorized users.

Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria.

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc/μl) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/μl). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ∼80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients.

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration

Our previous reports indicate that ligand-induced αVβ3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca2+ to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVβ3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVβ3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca2+, hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVβ3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca2+ entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production

Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases.

The Induction of Inhibitory Pathways in Dendritic Cells May Hamper the Efficient Activation of Anti-Leukemia T Cells within Chemotherapy-Induced Immunogenic Cell Death

BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) is still poor due to the lack of novel and effective therapies. In different malignancies including AML, some chemotherapy agents, such as daunorubicin (DNR) but not cytarabine (Ara-C), activate the immune response via the cross-priming of anti-tumor T cells by dendritic cells (DCs). Such process, known as immunogenic cell death (ICD), is characterized by intracellular and pericellular modifications of tumor cells, such as the cell surface translocation of calreticulin (CRT) and heat shock proteins 70/90 (HSPs 70/90), the extracellular release of ATP and pro-inflammatory factor HMGB1. Alongside with ICD, chemotherapy is known to induce inflammatory modifications within the tumor microenvironment, which may also elicit immunosuppressive pathways. In particular, DCs may be driven to acquire tolerogenic features, which may ultimately affect anti-tumor T-cell responses. In this study, we characterize ICD in AML to evaluate the involvement of some DC-related inhibitory pathways, such as the expression of indoleamine-2,3-dioxygenase 1 (IDO1) and the activation of PD-L1/PD-1 axis.METHODS: AML patients were analyzed at diagnosis.Before and after DNR-based chemotherapy, patient-derived T cells were extensively characterized by FACS and analyzed for their capacity to produce IFN-γ in response to autologous blasts. The AML cell line HL-60 and primary AML cells were then exposed, in vitro, to different drugs, including DNR and, as control drug, Ara-C. Dying cells were tested for the surface expression of CRT and HSPs 70/90, the release of HMGB1 and ATP. Functionally, immature DCs generated from healthy donors were pulsed with DNR-treated AML cells. Then, loaded DCs were tested for the expression of maturation-associated markers and of inhibitory pathways, such as IDO1 and PD-L1 and used to stimulate autologous CD3+ T cells. After co-culture, autologous healthy donor T cells were analyzed for IFN-g production, PD-1 expression and Tregs induction. A mouse model was set up to investigate in vivo the mechanism(s) underlying ICD in AML. The murine myelomonocytic leukemia cell line WEHI was transfected with luciferase PmeLUC probe, inoculated subcutaneously into BALB/c mice and used to measure in vivo ATP release after chemotherapy. Tumor-infiltrating T cells and DCs were characterized and correlated with ATP release.RESULTS: DNR treatment induced ICD-related modifications in both AML cell lines and primary blasts, including CRT, HSP70 and HSP90 exposure on cell surface, HMGB1 release from nucleus to cytoplasm and supernatant increase of ATP. Ex vivo, T-cell monitoring of DNR-treated AML patients displayed an increase in leukemia-specific IFN-g-producing CD4+ and CD8+ T cells in 20/28 evaluated patients. However, FACS analysis of CD8+ effector T cells emerging after chemotherapy showed a significant up-regulation of exhaustion marker such as LAG3 and PD-1, which paralleled with their reduced ability to produce active effector molecules, such as perforin and granzyme. Moreover, an increase of circulating Tregs was observed after DNR-based chemotherapy. In vitro, loading of chemotherapy-treated AML cells into DCs resulted not only in the induction of a maturation phenotype, but also in over-expression of inhibitory pathways, such as IDO1 and PD-L1. The silencing of IDO1 increased the capacity of DCs loaded with DNR-treated AML cells to induce leukemia-specific IFN-γ production by CD4+ and CD8+ T cells. In vivo, DNR therapy of mice inoculated with established murine AML cell line resulted in increased ATP release. Similarly to ex vivo and in vitro results, tumor-infiltrating DCs showed an increase in maturation status. Moreover, CD4+ and CD8+ T cells had increased IFN-γ production, but showed an exhausted phenotype.CONCLUSIONS: Our data confirm that chemotherapy-induced ICD may be active in AML and results in increased leukemia-specific T-cell immune response. However, a deep, ex vivo, in vitro and in vivo characterization of chemotherapy-induced T cells demonstrated an exhausted phenotype, which may be the result of the inhibitory pathways induction in DCs, such as IDO and PD-L1. The present data suggest that combination of chemotherapy with inhibitors of IDO1 and PD-L1 may represent an interesting approach to potentiate the immunogenic effect of chemotherapy, thus resulting in increased anti-leukemia immune response. DisclosuresCavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Electrical properties of purinergic transmission in smooth muscle of the guinea-pig prostate.

Prostatic smooth muscle develops spontaneous myogenic tone which is modulated by autonomic neuromuscular transmission. This study aimed to investigate the role of purinergic transmission in regulating electrical activity of prostate smooth muscle and whether its contribution may be altered with age. Intracellular recordings were simultaneously made with isometric tension recordings in smooth muscle preparations of the guinea-pig prostate. Immunostaining for P2X1 receptors on whole mount preparations was also performed. In prostate preparations which generated spontaneous slow waves, electrical field stimulation (EFS)-evoked excitatory junction potentials (EJPs) which were abolished by guanethidine (10 μM), α-β-methylene ATP (10 μM) or pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS, 10 μM) but not phentolamine (1 μM). Consistently, immunostaining revealed the expression of P2X1 receptors on prostatic smooth muscle. EJPs themselves did not cause contractions, but EJPs could sum to trigger a slow wave and associated contraction. Yohimbine (1 μM) and 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM) but not propranolol (1 μM) potentiated EJPs. Although properties of EJPs were not different between young and aging guinea-pig prostates, ectoATPase inhibitor ARL 67156 (100 μM) augmented EJP amplitudes by 64.2 ± 29.6% in aging animals, compared to 22.1 ± 19.9% in young animals. These results suggest that ATP released from sympathetic nerves acts on P2X1 purinoceptors located on prostate smooth muscle to evoke EJPs, while pre-junctional α2-adrenergic and adenosine A2 receptors may play a role in preventing excessive transmitter release. Age-related up-regulation of enzymatic ATP breakdown may be a compensatory mechanism for the enhanced purinergic transmission which would cause hypercontractility arising from increased ATP release in older animals.

Protease-Activated Receptor-2 Up-Regulates Transient Receptor Potential Vanilloid 4 Function in Mouse Esophageal Keratinocyte.

The reflux of pancreatic-duodenal fluids is implicated in the pathophysiology of proton-pump inhibitor-resistant gastroesophageal reflux disease (GERD). Protease-activated receptor-2 (PAR-2) is activated by proteases, the pancreatic enzyme, trypsin, and the activated PAR-2 enhances transient receptor potential vanilloid 4 (TRPV4) function in neurons. TRPV4 stimulates ATP exocytosis in conjunction with the vesicular nucleotide transporter, which mediates mechano-transduction and vagal stimulation. The aim of the present study was to verify whether the activated PAR-2 up-regulates TRPV4 function in mouse esophageal keratinocytes, which may link to the pathophysiology in PPI-resistant GERD. TRPV4 and PAR-2 expressions were detected by RT-PCR, immunostaining, and western blotting in mouse esophageal keratinocytes. The functional response of TRPV4 to esophageal keratinocytes was analyzed using a Ca(2+) imaging system. Cellular ATP release was examined by luciferase-luciferin reaction. TRPV4 phosphorylation was studied by immunoprecipitation and western blotting. PAR-2 and TRPV4 mRNAs and proteins were expressed in esophageal keratinocytes. Pre-treatment with trypsin significantly increased the responses to TRPV4 activator in esophageal keratinocytes, probably via the phosphorylation of serine residue of TRPV4 by protein kinase C and resulted in cellular ATP release from the cells. Activated PAR-2 with trypsin exposure up-regulated TRPV4 function and increased ATP release in mouse esophageal keratinocytes. This mechanism might be related to the pathophysiology of GERD, especially non-erosive GERD.

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

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

E-NTPDase1/CD39 modulates renin release from heart mast cells during ischemia/reperfusion: a novel cardioprotective role.

Ischemia/reperfusion (I/R) elicits renin release from cardiac mast cells (MC), thus activating a local renin-angiotensin system (RAS), culminating in ventricular fibrillation. We hypothesized that in I/R, neurogenic ATP could degranulate juxtaposed MC and that ecto-nucleoside triphosphate diphosphohydrolase 1/CD39 (CD39) on MC membrane could modulate ATP-induced renin release. We report that pharmacological inhibition of CD39 in a cultured human mastocytoma cell line (HMC-1) and murine bone marrow-derived MC with ARL67156 (100 µM) increased ATP-induced renin release (≥2-fold), whereas purinergic P2X7 receptors (P2X7R) blockade with A740003 (3 µM) prevented it. Likewise, CD39 RNA silencing in HMC-1 increased ATP-induced renin release (≥2-fold), whereas CD39 overexpression prevented it. Acetaldehyde, an I/R product (300 µM), elicited an 80% increase in ATP release from HMC-1, in turn, causing an autocrine 20% increase in renin release. This effect was inhibited or potentiated when CD39 was overexpressed or silenced, respectively. Moreover, P2X7R silencing prevented ATP- and acetaldehyde-induced renin release. I/R-induced RAS activation in ex vivo murine hearts, characterized by renin and norepinephrine overflow and ventricular fibrillation, was potentiated (∼2-fold) by CD39 inhibition, an effect prevented by P2X7R blockade. Our data indicate that by regulating ATP availability at the MC surface, CD39 modulates local renin release and thus, RAS activation, ultimately exerting a cardioprotective effect.

Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons

Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1Hz applied 30min later. This induction of DP was inhibited by application of either 100nM adenosine or 0.5nM N6-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice.

P2Y2 R activation by nucleotides promotes skin wound-healing process.

P2Y2 R has been shown to be upregulated in a variety of tissues in response to stress or injury and to mediate tissue regeneration through its ability to activate multiple signalling pathways. This study aimed to investigate the role of P2Y2 R in the wound-healing process and the mechanisms by which P2Y2 R activation promotes wound healing in fibroblasts. The role of P2Y2 R in skin wound healing was examined using a full-thickness skin wound model in wildtype (WT) and P2Y2 R(-/-) mice and an in vitro scratch wound model in control or P2Y2 R siRNA-transfected fibroblasts. WT mice showed significantly decreased wound size compared with P2Y2 R(-/-) mice at day 14 post-wounding, and immunohistochemical analysis showed that a proliferation marker Ki67 and extracellular matrix (ECM)-related proteins VEGF, collagen I, fibronectin and α-SMA were overexpressed in WT mice, which were reduced in P2Y2 R(-/-) mice. Scratch-wounded fibroblasts increased ATP release, which peaked at 5 min. In addition, scratch wounding increased the level of P2Y2 R mRNA. Activation of P2Y2 R by ATP or UTP enhanced proliferation and migration of fibroblasts in in vitro scratch wound assays and were blocked by P2Y2 R siRNA. Finally, ATP or UTP also increased the levels of ECM-related proteins through the activation of P2Y2 R in fibroblasts. This study suggests that P2Y2 R may be a potential therapeutic target to promote wound healing in chronic wound diseases.

Cav1.1 controls ATP release to elicit gene expression in adult muscle fibers (1164.12)

In adult muscle fibers, Cav1.1 acts as voltage sensor for both excitation‐contraction coupling and the activation of a signaling cascade that regulates gene expression. We have shown that ATP is released through pannexin‐1 channels after electrical stimulation at 20 Hz, having a key role in the induction of transcriptional changes related to fast‐to‐slow muscle fiber phenotype transition. Myotubes lacking the Cav1.1α1 subunit displayed almost no ATP release after electrical stimulation. ATP release and gene transcription was also altered in adult muscle fibers knock‐down for Cav1.1α1.In dystrophic fibers, there was also an increase in basal ATP release, but a default in the stimulation‐dependent increase in ATP release and transcription activation observed in control fibers. This can be interpreted as uncoupling between electrical stimulation and ATP release in dystrophic muscles.All these data suggest that Cav1.1 controls ATP release trough Pannexin‐1 channels, activating it after low stimulation frequencies and blocking ATP release during resting conditions. The loss of this control perturbs the normal transcriptional response to electrical activity of adult muscle fibers and may play a role in the etiology of muscular dystrophy.Grant Funding Source: ACT 1111, FONDECYT 1110467, UINICIA 0212M, CNRS‐CONICYT EDC24748

Pannexin 1‐dependent ATP release from venous endothelium promotes acute vascular inflammation (669.8)

Tumor necrosis factor α (TNFα) activates endothelial cells (EC) promoting EC‐leukocyte interactions in the venous circulation which is mediated, in part, by purinergic signaling. However, it is currently not known if ECs release ATP to TNFα. Employing in vitro ATP release assays, we report that TNFα significantly increased ATP release from venous ECs (Human Umbilical and Saphenous) which was not observed in arterial ECs (Human Coronary and Aortic). ATP release was inhibited by WP9QY, a peptide antagonist of the TNF receptor 1 (TNFR1), indicating receptor activation in the initiation of this event. Pharmacological inhibition of Pannexin 1 (Panx1) channels in venous ECs with CBX or 10panx1 and siRNA knockdown of Panx1 significantly inhibited ATP release, whereas inhibition of connexin hemichannels, CALHM1 channels and exocytosis had no effect. This indicates that Panx1 channels are the conduit for ATP liberation. Additionally, we show that Src family kinases mediate Panx1 channel activation, as the Src inhibitor PP2 significantly reduced TNFα‐induced ATP release. Functionally, both Panx1 inhibition and degradation of extracellular ATP attenuated leukocyte‐venous EC interactions to TNFα stimulation. Utilizing ex vivo cannulated mesenteric venules, luminal perfusion of TNFα significantly increased ATP release into the perfusate, supporting a role for TNFα‐mediated ATP release in intact venules. Finally, we developed an EC‐specific, inducible Panx1 knockout mouse (VEcadCreERT2+/Panx1fl/fl) to determine the role of Panx1 in vascular inflammation in vivo. In conclusion, TNFα activates Panx1 channels in venous ECs inducing ATP release to promote leukocyte‐EC interactions.Grant Funding Source: Supported by NIH, AHA

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.

Feed-forward inhibition of CD73 and upregulation of adenosine deaminase contribute to the loss of adenosine neuromodulation in postinflammatory ileitis.

Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders.