Ectonucleotidase Inhibitor Research Articles

Assessment of ATP metabolism to adenosine by ecto-nucleotidases carried by tumor-derived small extracellular vesicles.

Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression.Here, the ATP pathway of ADO production (ATP ADP AMP ADO) by ecto-nucleotidases carried on the sEV surface was evaluated by a method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates for enzymatic activity. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL).Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP. MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation by both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by ecto-nucleotidases located on the sEV surface.The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of ecto-nucleotidase activity primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.

Differential effects of purines and prostaglandins on hypoxia induced dilatation of porcine retinal vessels at different branching level ex vivo

The metabolic pathways leading from hypoxia to retinal vasodilatation can involve effects of both purines and prostaglandins, but the effects of these compounds at different vascular branching levels are unknown. The purpose of the present study was to investigate differential effects of purines and prostaglandins in hypoxia-induced dilatation of retinal arterioles, precapillary arterioles and capillaries ex vivo.Porcine hemiretinas were mounted in a tissue chamber while monitoring temperature, pH, and oxygen tension. The effect of hypoxia on the diameter of larger arterioles, precapillary arterioles and capillaries was studied in the presence of the ecto-nucleotidase inhibitor AOPCP, the nonselective P2 purinoreceptor antagonist PPADS, the A2B adenosine receptor antagonist MRS 1754, the A3 adenosine receptor antagonist MRS 1523, the EP1 receptor antagonist SC-19220, the EP2 receptor antagonist PF-04418948, the EP3 receptor antagonist L-798,106, the EP4 receptor antagonist L-161-982, the prostaglandin synthesis inhibitor ibuprofen, and ibuprofen combined with AOPCP or ATP.Hypoxia-induced dilatation in arterioles was reduced by the A2B adenosine receptor antagonist (p < 0.01) and increased by the EP2 and the EP3 receptor antagonists (p < 0.01 for both comparisons). In precapillary arterioles the dilatation was reduced by the EP2 receptor antagonist (p < 0.04) and increased by the EP1 receptor antagonist (p < 0.03), whereas in capillaries the dilatation was increased by both the A3 adenosine receptor antagonist (p < 0.01), by ibuprofen in combination with the unspecific ecto-nucleotidase inhibitor AOPCP (p = 0.04) and by the prostaglandin EP3 receptor antagonist.Hypoxia-induced dilatation of retinal vessels is influenced by adenosine A2B and A3 receptors, and by the prostaglandin EP1, EP2 and EP3 receptors. The effects mediated by these receptors differ at different branching levels of the resistance vessels.

Indoxyl sulfate decreases uridine adenosine tetraphosphate-induced contraction in rat renal artery.

The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat's renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K+-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.

Regulation of blood vessels by ATP in the ventral medullary surface in a rat model of Parkinson’s disease

Parkinson’s disease (PD) patients often experience impairment of autonomic and respiratory functions. These include conditions such as orthostatic hypotension and sleep apnea, which are highly correlated with dysfunctional central chemoreception. Blood flow is a fundamental determinant of tissue CO2/H+, yet the extent to which blood flow regulation within chemoreceptor regions contributes to respiratory behavior during neurological disease remains unknown. Here, we tested the hypothesis that 6-hydroxydopamine injection to inducing a known model of PD results in dysfunctional vascular homeostasis, biochemical dysregulation, and glial morphology of the ventral medullary surface (VMS). We show that hypercapnia (FiCO2 = 10%) induced elevated VMS pial vessel constriction in PD animals through a P2-receptor dependent mechanism. Similarly, we found a greater CO2-induced vascular constriction after ARL67156 (an ectonucleotidase inhibitor) in control and PD-induced animals. In addition, we also report that weighted gene correlational network analysis of the proteomic data showed a protein expression module differentially represented between both groups. This module showed that gene ontology enrichment for components of the ATP machinery were reduced in our PD-model compared to control animals. Altogether, our data indicate that dysfunction in purinergic signaling, potentially through altered ATP bioavailability in the VMS region, may compromise the RTN neuroglial vascular unit in a PD animal model.

Delivery of an ectonucleotidase inhibitor with ROS-responsive nanoparticles overcomes adenosine-mediated cancer immunosuppression.

Tumor evasion of immune destruction is associated with the production of immunosuppressive adenosine in the tumor microenvironment (TME). Anticancer therapies can trigger adenosine triphosphate (ATP) release from tumor cells, causing rapid formation of adenosine by the ectonucleotidases CD39 and CD73, thereafter exacerbating immunosuppression in the TME. The goal of this study was to develop an approach to facilitate cancer therapy-induced immunogenic cell death including ATP release and to limit ATP degradation into adenosine, in order to achieve durable antitumor immune response. Our approach was to construct reactive oxygen species (ROS)-producing nanoparticles that carry an ectonucleotidase inhibitor ARL67156 by electronic interaction and phenylboronic ester. Upon near-infrared irradiation, nanoparticle-produced ROS induced ATP release from MOC1 cancer cells in vitro and triggered the cleavage of phenylboronic ester, facilitating the release of ARL67156 from the nanoparticles. ARL67156 prevented conversion of ATP to adenosine and enhanced anticancer immunity in an MOC1-based coculture model. We tested this approach in mouse tumor models. Nanoparticle-based ROS-responsive drug delivery reprogramed the immunogenic landscape in tumors, eliciting tumor-specific T cell responses and tumor regression, conferring long-term survival in mouse models. We demonstrated that TME reprograming sets the stage for response to anti-programmed cell death protein 1 (PD1) immunotherapy, and the combination resulted in tumor regression in a 4T1 breast cancer mouse model that was resistant to PD1 blockade. Furthermore, our approach also induced immunological effects in patient-derived organotypic tumor spheroid model, suggesting potential translation of our nanoparticle approach for treating human cancers.

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.

CD73 expression in myeloid-derived suppressor cells is correlated with clinical stages in head and neck squamous cell carcinomas.

BackgroundEcto-5'-nucleotidase (cluster of differentiation 73/CD73) is an ectonucleotidase that is being evaluated as a biomarker for the diagnosis and prognosis of various types of cancer. However, the clinicopathological relationship between CD73 expression in monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSC (PMN-MDSCs) in head and neck squamous cell carcinomas (HNSCCs) is not clear. Understanding the phenotypic and functional characteristics of human CD73+ MDSCs in the tumor microenvironment could help elucidate the roles of these cells in the ontogeny, spread, and treatment of solid cancer.MethodsIn the present study, we first analyzed the expression percentage of human M-MDSCs and PMN-MDSCs subsets circulating in peripheral blood of patients with head and neck tumors originated in nasopharynx, oropharynx, oropharynx and larynx. To identify the correlation between phenotypic characteristics of MDSCs and clinical stages in HNSCC, we extended the study by analyzing the percentage, CD73 phenotype and immunosuppressive function of MDSCs and the correlation with the clinical parameters. Moreover, we compare the functions of both M-MDSCs and PMN-MDSCs blunts T-cell function in an ectonucleotidase-dependent manner.ResultsOur study revealed that PMN-MDSCs were significantly increased in HNSCC patients, contributing to MDSC-mediated T cell immune suppression. Our results indicated that PMN-MDSCs comprised the majority of MDSCs participating in anticancer immunosuppression. The increase in PMN-MDSCs was directly correlated with the clinical stages of HNSCC. Levels of CD73 were increased in PMN-MDSCs and were correlated with the clinical stages of HNSCC. The ectonucleotidase inhibitor adenosine 5'-(α,β-methylene)diphosphate (APCP) decreased its suppression towards T cell proliferation. Ectonucleotidase inhibitors are promising candidates for the treatment of HNSCC.ConclusionsThese studies demonstrate the expansion of PMN-MDSCs correlated with expression of CD73 and increasing clinical stages in HNSCC. These CD73+ PMN-MDSCs contributes to T cell immune suppression activity in HNSCC patients. Using ectonucleotidase inhibitors is a promising rationale for PMN-MDSCs in future clinical development of immunotherapy in human HNSCC cancer.

Chronic fructose renders pancreatic β-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic β-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human β-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the β-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in β-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve β-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic β-cells mediating fructose-induced hyper-responsiveness.

Purinergic Signaling Modulates Survival/Proliferation of Human Dental Pulp Stem Cells

Human dental pulp stem cells (hDPSCs) reside in postnatal dental pulp and exhibit the potential to differentiate into odontoblasts as well as neurons. However, the intercellular signaling niches necessary for hDPSC survival and self-renewal remain largely unknown. The objective of this study is to demonstrate the existence of intercellular purinergic signaling in hDPSCs and to assess the impact of purinergic signaling on hDPSC survival and proliferation. hDPSCs were isolated from extracted third molars and cultured in minimum essential medium. To demonstrate responsiveness to ATP application and inhibitions by purinergic receptor antagonists, whole cell patch-clamp recordings of ATP-induced currents were recorded from cultured hDPSCs. Immunofluorescence and enzymatic histochemistry staining were performed to assess purinergic receptor expression and ectonucleotidase activity in hDPSCs, respectively. To determine the effects of purinergic signaling on hDPSC, purinergic receptor antagonists and an ectonucleotidase inhibitor were applied in culture medium, and hDPSC survival and proliferation were assessed with DAPI staining and Ki67 immunofluorescence staining, respectively. We demonstrated that ATP application induced inward currents in hDPSCs. P2X and P2Y receptors are involved in the generation of ATP-induced inward currents. We also detected expression of NTPDase3 and ectonucleotidase activity in hDPSCs. We further demonstrated that purinergic receptors were tonically activated in hDPSCs and that inhibition of ectonucleotidase activity enhanced ATP-induced inward currents. Furthermore, we found that blocking P2Y and P2X receptors reduced—and inhibition of ecto-ATPase activity enhanced—the survival and proliferation of hDPSCs, while blocking P2X receptors alone affected only hDPSC proliferation. Autocrine/paracrine purinergic signaling is essential for hDPSC survival and proliferation. These results reveal potential targets to manipulate hDPSCs to promote tooth/dental pulp repair and regeneration.

Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats

The brain regulates breathing in response to changes in tissue CO2/H+ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO2/H+-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7–10% CO2 (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO2, suggesting a portion of ATP released in the RTN during high CO2/H+ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Extracellular adenosine 5’-triphosphate concentrations changes in rat spinal cord associated with the activation of urinary bladder afferents. A microdialysis study

ABSTRACTObjective To determine adenosine 5’-triphosphate levels in the interstice of spinal cord L6-S1 segment, under basal conditions or during mechanical and chemical activation of urinary bladder afferents.Methods A microdialysis probe was transversally implanted in the dorsal half of spinal cord L6-S1 segment in female rats. Microdialysate was collected at 15 minutes intervals during 135 minutes, in anesthetized animals. Adenosine 5’-triphosphate concentrations were determined with a bioluminescent assay. In one group of animals (n=7) microdialysate samples were obtained with an empty bladder during a 10-minutes bladder distension to 20 or 40cmH2O with either saline, saline with acetic acid or saline with capsaicin. In another group of animals (n=6) bladder distention was performed and the microdialysis solution contained the ectonucleotidase inhibitor ARL 67156.Results Basal extracellular adenosine triphosphate levels were 110.9±35.34fmol/15 minutes, (mean±SEM, n=13), and bladder distention was associated with a significant increase in adenosine 5’-triphosphate levels which was not observed after bladder distention with saline solution containing capsaicin (10µM). Microdialysis with solution containing ARL 67156 (1mM) was associated with significantly higher extracellular adenosine 5’-triphosphate levels and no further increase in adenosine 5’-triphosphate was observed during bladder distension.Conclusion Adenosine 5’-triphosphate was present in the interstice of L6-S1 spinal cord segments, was degraded by ectonucleotidase, and its concentration increased following the activation of bladder mechanosensitive but not of the chemosensitive afferents fibers. Adenosine 5’-triphosphate may originate either from the central endings of bladder mechanosensitive primary afferent neurons, or most likely from intrinsic spinal neurons, or glial cells and its release appears to be modulated by capsaicin activated bladder primary afferent or by adenosine 5’-triphosphate itself.

Central Role of P2Y 6 UDP Receptor in Arteriolar Myogenic Tone

Objective— Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results— We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X 1 and P2Y 6 was dominant. P2Y 6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6 −/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y 6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6 −/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y 6 and P2Y 2 receptors contributed to calcium mobilization, P2Y 6 activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43 +/− and P2rx7 −/− mesenteric resistance arteries. Conclusions— Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y 6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Postsynaptic Potentiation in Mouse Motor Synapses Induced by ATP Accumulation in Synaptic Cleft.

In mouse motor synapses, a non-selective purinoceptor antagonist suramin increased the quantum content of endplate potentials (EPP) without changing the time course of synaptic potentials. An ectonucleotidase inhibitor ARL 67156 had no effect on the amplitude and quantum content of EPP and miniature endplate potentials (mEPP) evoked by single stimuli, but significantly prolonged their duration. Long-term high-frequency stimulation of the nerve in the presence of ARL 67156 persistently increased the amplitude and duration of EPP during the train of impulses, but did not change their quantum content. ATP-γ-S, a non-hydrolyzed ATP analogue, significantly increased the amplitudes and prolonged the rising and falling phases of EPP and mEPP. The ATP-induced postsynaptic potentiation in neuromuscular transmission can result from the increase in ATP content and its longer presence in the synaptic cleft.

Stimulation of Glia Reveals Modulation of Mammalian Spinal Motor Networks by Adenosine.

Despite considerable evidence that glia can release modulators to influence the excitability of neighbouring neurons, the importance of gliotransmission for the operation of neural networks and in shaping behaviour remains controversial. Here we characterise the contribution of glia to the modulation of the mammalian spinal central pattern generator for locomotion, the output of which is directly relatable to a defined behaviour. Glia were stimulated by specific activation of protease-activated receptor-1 (PAR1), an endogenous G-protein coupled receptor preferentially expressed by spinal glia during ongoing activity of the spinal central pattern generator for locomotion. Selective activation of PAR1 by the agonist TFLLR resulted in a reversible reduction in the frequency of locomotor-related bursting recorded from ventral roots of spinal cord preparations isolated from neonatal mice. In the presence of the gliotoxins methionine sulfoximine or fluoroacetate, TFLLR had no effect, confirming the specificity of PAR1 activation to glia. The modulation of burst frequency upon PAR1 activation was blocked by the non-selective adenosine-receptor antagonist theophylline and by the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, but not by the A2A-receptor antagonist SCH5826, indicating production of extracellular adenosine upon glial stimulation, followed by A1-receptor mediated inhibition of neuronal activity. Modulation of network output following glial stimulation was also blocked by the ectonucleotidase inhibitor ARL67156, indicating glial release of ATP and its subsequent degradation to adenosine rather than direct release of adenosine. Glial stimulation had no effect on rhythmic activity recorded following blockade of inhibitory transmission, suggesting that glial cell-derived adenosine acts via inhibitory circuit components to modulate locomotor-related output. Finally, the modulation of network output by endogenous adenosine was found to scale with the frequency of network activity, implying activity-dependent release of adenosine. Together, these data indicate that glia play an active role in the modulation of mammalian locomotor networks, providing negative feedback control that may stabilise network activity.

Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion

Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

Uridine adenosine tetraphosphate is a novel vasodilator in the coronary microcirculation which acts through purinergic P1 but not P2 receptors

Uridine adenosine tetraphosphate (Up4A) has been identified as an endothelium-derived contracting factor, which acts through purinergic P2X and P2Y receptors. Since the coronary vascular actions of Up4A are unknown, we investigated the vasoactive profile of Up4A in coronary microvessels, and studied the involvement of purinergic receptor subtypes. Studies were performed in isolated porcine coronary small arteries (diameter∼250μm), with and without endothelial denudation, mounted on a Mulvany wire myograph. Purinergic receptor expression was assessed by real-time PCR. Up4A (10−9–10−5M) failed to induce contraction at basal tone, but produced concentration-dependent vasorelaxation in precontracted microvessels. Up4A was slightly less potent than adenosine, ATP, and ADP in producing vasorelaxation, but significantly more potent than UTP and UDP. mRNA expression of P2X4, P2Y1, P2Y2, P2Y4, P2Y6 and A2A, but not P2X1, receptors was observed. Up4A-induced vasodilation was unaffected by non-selective P2 receptor antagonist PPADS, P2X1 antagonist MRS2159, P2Y1 antagonist MRS2179 and P2Y6 antagonist MRS2578, but was markedly attenuated by non-selective P1 receptor antagonist 8PT and A2A antagonist SCH58261. Up4A-induced vasodilation was not affected by ectonucleotidase inhibitor ARL67156, suggesting that A2A stimulation was not the result of Up4A breakdown to adenosine. Up4A-induced vasodilation was blunted in denuded vessels; additional A2A receptor blockade further attenuated Up4A-induced vasodilation, suggesting that A2A receptor-mediated vasodilation is only partly endothelium-dependent. In conclusion, Up4A exerts a vasodilator rather than a vasoconstrictor influence in coronary microvessels, which is mediated via A2A receptors and is partly endothelium-dependent.

Autocrine regulation of γ-irradiation-induced DNA damage response via extracellular nucleotides-mediated activation of P2Y6 and P2Y12 receptors

A key component of the response to DNA damage caused by ionizing radiation is DNA repair. Release of extracellular nucleotides, such as ATP, from cells plays a role in signaling via P2 receptors. We show here that release of ATP, followed by activation of P2Y receptors, is involved in the response to γ-irradiation-induced DNA damage. Formation of phosphorylated histone variant H2AX (γH2AX) foci, which are induced in nuclei by DNA damage and contribute to accumulation of DNA-repair factors, was increased at 1–3h after γ-ray irradiation (2.0Gy) of human lung cancer A549 cells. Focus formation was suppressed by pre-treatment with the ecto-nucleotidase apyrase. Pre-treatment with ecto-nucleotidase inhibitor ARL67156 or post-treatment with ATP or UTP facilitated induction of γH2AX, indicating that extracellular nucleotides play a role in induction of γH2AX foci. Next, we examined the effect of P2 receptor inhibitors on activation of ataxia telangiectasia mutated (ATM; a protein kinase) and accumulation of 53BP1 (a DNA repair factor), both of which are important for DNA repair, at DNA damage sites. P2Y6 receptor antagonist MRS2578, P2Y12 receptor antagonist clopidogrel, and P2X7 receptor antagonists A438079 and oxATP significantly inhibited these processes. Release of ATP was detected within 2.5min after irradiation, but was blocked by A438079. Activation of ATM and accumulation of 53BP1 were decreased in P2Y6 or P2Y12 receptor-knockdown cells. We conclude that autocrine/paracrine signaling through P2X7-dependent ATP release and activation of P2Y6 and P2Y12 receptors serves to amplify the cellular response to DNA damage caused by γ-irradiation.

Inhibition of ectonucleotidase with ARL67156 prevents the development of calcific aortic valve disease in warfarin-treated rats

Calcific aortic valve disease is the most common heart valve disorder. So far, there is no medical treatment for calcific aortic valve disease. The expression of ectonucleotidases, which metabolize nucleotides into phosphate products, may influence the calcification of the aortic valve. In this study, we investigated if the administration of an ectonucleotidase inhibitor, ARL67156 (6-N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt), may prevent the calcification of the aortic valve in the warfarin-induced mineralization rat model. Male Wistar rats were treated with warfarin or warfarin+ARL67156 for 28 days. All rats had comprehensive Doppler-echocardiographic studies at 28 day. A gene profiling of ectonucleotidases expressed in aortas of rats was documented by quantitative real-time PCR. The amount of calcium was determined by quantitative method and von Kossa staining. Ex vivo cultures of rat aortas were also used to further assess the effect of ARL67156 on the calcifying process and Akt signaling. Mineralization of the aorta/aortic valve was documented in warfarin-treated rats and was accompanied by the development of aortic stenosis. These changes were paralleled by an increased of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). Administration of the ectonucleotidase inhibitor, ARL67156 prevented the development of aortic stenosis by lowering the level of apoptosis and mineralization of the aortic valve/aorta. In addition, ARL67156 normalized the level of pAkt, an important kinase involved in the survival pathway. Inhibition of ectonucleotidase activity prevented the development of calcific aortic valve disease in a rat model. On that account, ectonucleotidase may represent a novel target in the treatment of calcific aortic valve disease.

AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

Adenosine and NO blockade decreases skeletal muscle interstitial pH during hypoxia

The present study examined the effects that blocking local adenosine and nitric oxide production has on skeletal muscle interstitial pH and H+ during hypoxia. Eight anesthetized rats had two microdialysis fibers inserted into the gastrocnemius muscle of each leg and perfused with Ringers (control) or 500 μM AOPCP (5′ectonucleotidase inhibitor) plus 10 mM L‐NAME (NOS inhibitor). Following baseline collections (20 min with saline, Baseline 1), all the probes were perfused with the both blockers for (20 min, Baseline 2) followed by 20 min of hypoxia (inhalation of 10.5% O2) and recovery. The perfusion of the blockers resulted in a decrease (P&lt;0.05) in skeletal muscle interstitial pH and a reciprocal increase (P&lt;0.05) in interstitial H+during normoxia (Baseline 2) and these changes were further exaggerated during hypoxia as well as recovery (Figures)1. These data clearly demonstrate that blocking local vasodilators most likely resulted in a decrease in local tissue blood flow and an accumulation of H+, which is further elevated under anaerobic conditions such as hypoxia.