The Purinergic Nature of Pseudoxanthoma Elasticum.

Pseudoxanthoma Elasticum (PXE) is an inherited disease characterized by elastic fiber calcification in the eyes, the skin and the cardiovascular system. PXE results from mutations in ABCC6 that encodes an ABC transporter primarily expressed in the liver and kidneys. It took nearly 15 years after identifying the gene to better understand the etiology of PXE. ABCC6 function facilitates the efflux of ATP, which is sequentially hydrolyzed by the ectonucleotidases ENPP1 and CD73 into pyrophosphate (PPi) and adenosine, both inhibitors of calcification. PXE, together with General Arterial Calcification of Infancy (GACI caused by ENPP1 mutations) as well as Calcification of Joints and Arteries (CALJA caused by NT5E/CD73 mutations), forms a disease continuum with overlapping phenotypes and shares steps of the same molecular pathway. The explanation of these phenotypes place ABCC6 as an upstream regulator of a purinergic pathway (ABCC6 → ENPP1 → CD73 → TNAP) that notably inhibits mineralization by maintaining a physiological Pi/PPi ratio in connective tissues. Based on a review of the literature and our recent experimental data, we suggest that PXE (and GACI/CALJA) be considered as an authentic "purinergic disease". In this article, we recapitulate the pathobiology of PXE and review molecular and physiological data showing that, beyond PPi deficiency and ectopic calcification, PXE is associated with wide and complex alterations of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1-/- male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1-/- mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1-/- mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1-/- mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1-/- mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female.

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5'-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1-/-) mice compared to wild type controls. The strongest responses were obtained with uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6-/- bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1-/-, and P2ry6-/- female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

Atrial Fibrillation Progression Is Associated with Cell Senescence Burden as Determined by p53 and p16 Expression.

BACKGROUND: Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear. AIMS: We investigated the link between AF and senescence by comparing the expression of senescence markers (p53 and p16), with prothrombotic and inflammatory proteins in right atrial appendages from patients in AF and sinus rhythm (SR). METHODS: The right atrial appendages of 147 patients undergoing open-heart surgery were harvested. Twenty-one non-valvular AF patients, including paroxysmal (PAF) or permanent AF (PmAF), were matched with 21 SR patients according to CHA2DS2-VASc score and treatment. Protein expression was assessed by tissue lysates Western blot analysis. RESULTS: The expression of p53, p16, and tissue factor (TF) was significantly increased in AF compared to SR (0.91 ± 0.31 vs. 0.58 ± 0.31, p = 0.001; 0.76 ± 0.32 vs. 0.35 ± 0.18, p = 0.0001; 0.88 ± 0.32 vs. 0.68 ± 0.29, p = 0.045, respectively). Expression of endothelial NO synthase (eNOS) was lower in AF (0.25 ± 0.15 vs. 0.35 ± 0.12, p = 0.023). There was a stepwise increase of p53, p16, TF, matrix metalloproteinase-9, and an eNOS progressive decrease between SR, PAF, and PmAF. AF was the only predictive factor of p53 and p16 elevation in multivariate analysis. Conclusions: The study brought new evidence indicating that AF progression is strongly related to human atrial senescence burden and points at a link between senescence, thrombogenicity, endothelial dysfunction and atrial remodeling.

Evidence of Cardiovascular Calcification and Fibrosis in Pseudoxanthoma Elasticum Mouse Models Subjected to DOCA-Salt Hypertension.

Pseudoxanthoma Elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Although arterial hypertension (AHT) has been reported in PXE patients, its impact on pathological manifestations has as yet been unexplored. We investigated the consequences of experimental AHT on Abcc6-/- PXE mouse models. Experimental AHT was induced by deoxycorticosterone acetate (DOCA-salt) in uni-nephrectomised mice. Blood pressure (BP) and vascular reactivity were monitored using tail-cuff plethysmography and myography respectively. Calcium content and fibrosis were assessed using colorimetry, Von Kossa and Sirius red staining respectively. The gene expression implicated in vascular biology was measured using quantitative polymerase chain reaction. DOCA-salt induced a matching rise in BP in Abcc6-/- and WT mice. Aortic ring contraction and relaxation in vitro were comparable. Calcium accumulated in the hearts of hypertensive Abcc6-/- mice along with significant fibrosis in the myocardium and aorta by contrast with the WT mice. In hypertensive Abcc6-/- mouse aortas, these results were corroborated by gene expression patterns favouring calcification, fibrosis and extracellular matrix remodelling. Abcc6 loss-of-function is associated with greater cardiovascular calcification and fibrosis in mice subjected to DOCA-Salt hypertension. These results suggest likely cardiovascular deterioration in PXE patients with AHT, necessitating diligent BP monitoring.

ABCC6 Deficiency Promotes Development of Randall Plaque.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disease caused by mutations in the ABCC6 gene that result in low pyrophosphate levels and subsequent progressive soft tissue calcifications. PXE mainly affects the skin, retina, and arteries. However, many patients with PXE experience kidney stones. We determined the prevalence of this pathology in patients with PXE and examined the possible underlying mechanisms in murine models. METHODS: We conducted a retrospective study in a large cohort of patients with PXE and analyzed urine samples and kidneys from Abcc6-/- mice at various ages. We used Yasue staining, scanning electron microscopy, electron microscopy coupled to electron energy loss spectroscopy, and Fourier transform infrared microspectroscopy to characterize kidney calcifications. RESULTS: Among 113 patients with PXE, 45 (40%) had a past medical history of kidney stones. Five of six computed tomography scans performed showed evidence of massive papillary calcifications (Randall plaques). Abcc6-/- mice spontaneously developed kidney interstitial apatite calcifications with aging. These calcifications appeared specifically at the tip of the papilla and formed Randall plaques similar to those observed in human kidneys. Compared with controls, Abcc6-/- mice had low urinary excretion of pyrophosphate. CONCLUSIONS: The frequency of kidney stones and probably, Randall plaque is extremely high in patients with PXE, and Abcc6-/- mice provide a new and useful model in which to study Randall plaque formation. Our findings also suggest that pyrophosphate administration should be evaluated for the prevention of Randall plaque and kidney stones.

Alteration of Extracellular Nucleotide Metabolism in Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a rare genetic condition primarily caused by hepatic ABCC6 transporter dysfunction. Most clinical manifestations of PXE are due to premature calcification of elastic fibers. However, the vascular impact of PXE is pleiotropic and remains ill defined. ABCC6 expression has recently been associated with cellular nucleotide export. We studied the impact of ABCC6 deficiency on blood levels of adenosine triphosphate and related metabolites and on soluble nucleotidase activities in PXE patients and Abcc6-/- mice. In addition, we investigated the expression of genes encoding ectocellular purinergic signaling proteins in mouse liver and aorta. Plasma adenosine triphosphate and pyrophosphate levels were significantly reduced in PXE patients and in Abcc6-/- mice, whereas adenosine concentration was not modified. Moreover, 5'-nucleotidase/CD73 activity was increased in the serum of PXE patients and Abcc6-/- mice. Consistent with alterations of purinergic signaling, the expression of genes involved in purine and phosphate transport/metabolism was dramatically modified in Abcc6-/- mouse aorta, with much less impact on the liver. ABCC6 deficiency causes impaired vascular homeostasis and tissue perfusion. Our findings suggest that these alterations are linked to changes in extracellular nucleotide metabolism that are remote from the liver. This opens new perspectives for the understanding of PXE pathophysiology.

Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension.

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

The ecto-ATPDase CD39 is involved in the acquisition of the immunoregulatory phenotype by M-CSF-macrophages and ovarian cancer tumor-associated macrophages: Regulatory role of IL-27.

Tumor-associated macrophages (TAM) are immunosuppressive cells that can massively accumulate in the tumor microenvironment. In patients with ovarian cancer, their density is correlated with poor prognosis. Targeting mediators that control the generation or the differentiation of immunoregulatory macrophages represents a therapeutic challenge to overcome tumor-associated immunosuppression. The ectonucleotidase CD39 hydrolyzes ATP into extracellular adenosine that exhibits potent immunosuppressive properties when signaling through the A2A adenosine receptor. We report here that CD14(+) CD163(+) TAM isolated from ovarian cancer patients and macrophages generated in vitro with M-CSF, express high levels of the membrane ectonucleotidase CD39 compared to classically activated macrophages. The CD39 inhibitor POM-1 and adenosine deaminase (ADA) diminished some of the immunosuppressive functions of CD14(high) CD163(high) CD39(high) macrophages, such as IL-10 secretion. We identified the cytokine IL-27, secreted by tumor-infiltrating neutrophils, located close to infiltrating CD163(+) macrophages, as a major rheostat of CD39 expression and consequently, on the acquisition of immunoregulatory properties by macrophages. Accordingly, the depletion of IL-27 downregulated CD39 and PD-L1 expression as well as IL-10 secretion by M-CSF-macrophages. Collectively, these data suggest that CD39, drived by IL-27 and CD115 ligands in ovarian cancer, maintains the immunosuppressive phenotype of TAM. This work brings new information on the acquisition of immunosuppressive properties by tumor-infiltrating macrophages.

Central Role of P2Y6 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 P2X1 and P2Y6 was dominant. P2Y6 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 P2Y6 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 P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 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 P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αßMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials.

Disseminated arterial calcification and enhanced myogenic response are associated with abcc6 deficiency in a mouse model of pseudoxanthoma elasticum.

OBJECTIVE: Pseudoxanthoma elasticum is an inherited metabolic disorder resulting from ABCC6 gene mutations. It is characterized by progressive calcification and fragmentation of elastic fibers in the skin, retina, and the arterial wall. Despite calcium accumulation in the arteries of patients with pseudoxanthoma elasticum, functional consequences remain unknown. In the present study, we investigated arterial structure and function in Abcc6(-/-) mice, a model of the human disease. APPROACH AND RESULTS: Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry. Expression of genes involved in osteochondrogenic differentiation was measured by polymerase chain reaction. Elastic arterial properties were evaluated by carotid echotracking. Vascular reactivity was evaluated using wire and pressure myography and remodeling using histomorphometry. Arterial calcium accumulation was 1.5- to 2-fold higher in Abcc6(-/-) than in wild-type mice. Calcium accumulated locally leading to punctuate pattern. Old Abcc6(-/-) arteries expressed markers of both osteogenic (Runx2, osteopontin) and chondrogenic lineage (Sox9, type II collagen). Abcc6(-/-) arteries displayed slight increase in arterial stiffness and vasoconstrictor tone in vitro tended to be higher in response to phenylephrine and thromboxane A2. Pressure-induced (myogenic) tone was significantly higher in Abcc6(-/-) arteries than in wild type. Arterial blood pressure was not significantly changed in Abcc6(-/-), despite higher variability. CONCLUSIONS: Scattered arterial calcium depositions are probably a result of osteochondrogenic transdifferentiation of vascular cells. Lower elasticity and increased myogenic tone without major changes in agonist-dependent contraction evidenced in aged Abcc6(-/-) mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis in the long term.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke.

AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.

Quantification of the calcification phenotype of Abcc6-deficient mice with microcomputed tomography.

Pseudoxanthoma elasticum in humans and dystrophic cardiac calcification in mice are heritable disorders characterized by dystrophic calcification of soft connective tissues related to the defective function of the ABCC6 (human)/Abcc6 (mouse) transporter. Of particular interest is the finding of calcified vibrissae in Abcc6(-/-) mice, which facilitates the study of dystrophic calcification by histological techniques. We aimed to determine whether mice prone to dystrophic cardiac calcification (C3H/HeOuJ and DBA/2J strains) presented similar vibrissae changes and to evaluate the value of microcomputed tomography to quantify the extent of mystacial vibrissae calcifications. These calcifications were absent in DBA/2J and C57BL/6J control mice. In both Abcc6(-/-) and C3H/HeOuJ mice, calcifications progressed in a caudal-rostral direction with aging. However, the calcification process was delayed in C3H/HeOuJ mice, indicating an incomplete expression of the calcification phenotype. We also found that the calcification process in the cephalic region was not limited to mystacial vibrissae but was also present in other periorbital sensorial vibrissae. The vibrissae calcification was circular and encompassed the medial region of the vibrissae capsule, adjacent to the ring and cavernous sinuses (the areas adjacent to blood and lymphatic vessels). Collectively, our findings confirm that Abcc6 acts as an inhibitor of spontaneous chronic mineralization and that microcomputed tomography is a valuable noninvasive tool for the assessment of the calcification phenotype in Abcc6-deficient mice.

Coexpression of ecto-5'-nucleotidase/CD73 with specific NTPDases differentially regulates adenosine formation in the rat liver.

Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5'-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high "inflammatory" (500 µM) and low "physiological" (1 µM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions.

Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion.

Extracellular nucleotides and adenosine regulate endocrine pancreatic functions such as insulin secretion by Langerhans islet ß-cells via the activation of specific P2 and P1 receptors. Membrane-bound ectonucleotidases regulate the local concentration of these ligands and consequently control the activation of their receptors. The objective of this study was to identify and localize the major ectonucleotidases, namely NTPDases and ecto-5'-nucleotidase, present in the endocrine pancreas. In addition, the potential implication of ecto-ATPase activity on insulin secretion was investigated in the rat ß-cell line INS-1 (832/13). The localization of ectonucleotidase activity and protein was carried out in situ by enzyme histochemistry and immunolocalization in mouse, rat, and human pancreas sections. NTPDase1 was localized in all blood vessels and acini, and NTPDase2 was localized in capillaries of Langerhans islets and in peripheral conjunctive tissue, whereas NTPDase3 was detected in all Langerhans islet cell types. Interestingly, among the mammalian species tested, ecto-5'-nucleotidase was present only in rat Langerhans islet cells, where it was coexpressed with NTPDase3. Notably, the inhibition of NTPDase3 activity by BG0136 and NF279 facilitated insulin release from INS-1 (832/13) cells under conditions of low glycemia, probably by affecting P2 receptor activation. NTPDase3 activity also regulated the inhibitory effect of exogenous ATP in the presence of a high glucose concentration most likely by controlling adenosine production. In conclusion, all pancreatic endocrine cells express NTPDase3 that was shown to modulate insulin secretion in rat INS-1 (832/13) ß-cells. Ecto-5'-nucleotidase is expressed in rat Langerhans islet cells but absent in human and mouse endocrine cells.

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation.

BACKGROUND AND PURPOSE: Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation. EXPERIMENTAL APPROACH: Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1(-/-) (lacking NTPDase1) mice. KEY RESULTS: The absence of NTPDase1 in Entpd1(-/-) mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1(+/+) and Entpd1(-/-) mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole.kg(-1) both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1(-/-) mice. EC(50) values in Entpd1(+/+) versus Entpd1(-/-) aortic rings were 14 microM versus 0.35 microM for ADP, and 29 microM versus 1 microM for ATP. In Entpd1(-/-) aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPbetaS (P2Y1) and ATPgammaS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice. CONCLUSIONS AND IMPLICATIONS: NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.

Epidermal growth factor receptor is an obligatory intermediate for oxytocin-induced cyclooxygenase 2 expression and prostaglandin F2 alpha production in bovine endometrial epithelial cells.

Oxytocin (OT) triggers the luteolytic pulses of prostaglandin F(2 alpha) (PGF(2 alpha)) from the endometrial epithelial cells in ruminants. We have proposed that the embryonic signal interferon-tau exerts its antiluteolytic effect by disrupting the OT signaling axis. Accordingly, we have attempted to define the signaling pathway of OT-induced PGF(2 alpha) production in the bovine endometrium using our newly characterized epithelial cell line (bEEL). OT receptor was coupled to the classical G alpha(q) pathway as evidenced by calcium release and activation of phospholipase C. Similarly, OT-induced PGF(2 alpha) production was mediated through the canonical ERK1/2 pathway. Because of the importance of receptor and nonreceptor tyrosine kinases in G protein-coupled receptor signaling, we studied the role of epidermal growth factor receptor (EGFR), c-Src, and phosphoinositide 3-kinase (PI3K) on OT-induced PGF(2 alpha) production in association with cyclooxygenase 2 (COX2) expression and ERK1/2 and Akt phosphorylation. The EGFR inhibitor AG1478 (10 microm) nearly abolished basal and OT-induced PGF(2 alpha) production and down-regulated COX2 expression and ERK1/2 phosphorylation. Because the transactivated EGFR can serve as a ligand for the signaling proteins with Src homology 2 (SH2) domain, we hypothesized a role for c-Src and PI3K in OT-induced PGF(2 alpha) production. Inhibitors of c-Src (PP2, 10 microm) and PI3K (LY294002, 25 microm) produced a significant decrease in OT-induced PGF(2 alpha) production and reduced COX2 expression. Also, PP2, but not LY294002, decreased OT-induced ERK1/2 phosphorylation. Because LY294002 did not affect ERK1/2 phosphorylation, but inhibited PGF(2 alpha) production and down-regulated COX2 expression, it is likely that the Akt pathway is also involved in PGF(2 alpha) production. Thus, EGFR may simultaneously activate c-Src and PI3K to amplify the OT signaling to increase the output of PGF(2 alpha) in bEEL cells.

NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse.

AIMS: Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. METHODS AND RESULTS: Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. CONCLUSION: NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.