Ectonucleoside Triphosphate Diphosphohydrolase Research Articles

Therapeutic Study of Cinnamic Acid Derivative for Oxidative Stress Ablation: The Computational and Experimental Answers.

This study aimed to examine the therapeutic activity of the cinnamic acid derivative KAD-7 (N'-(2,4-dichlorobenzylidene)-3-(4-methoxyphenyl) acrylohydrazide) on Fe2+-induced oxidative hepatic injury via experimental and computational models. In addition, the role of ATPase and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) in the coordination of cellular signals is speculated upon to proffer suitable therapeutics for metabolic stress disorder upon their inhibition. While we know little about therapeutics with flexible dual inhibitors for these protein targets, this study was designed to screen KAD-7's (N'-(2,4-dichlorobenzylidene)-3-(4-methoxyphenyl) acrylohydrazide) inhibitory potential for both protein targets. We induced oxidative hepatic damage via the incubation of hepatic tissue supernatant with 0.1 mM FeSO4 for 30 min at 37 °C. We achieved the treatment by incubating the hepatic tissues with KAD-7 under the same conditions. The catalase (CAT), glutathione (GSH), malondialdehyde (MDA), ATPase, and ENTPDase activity were all measured in the tissues. We predicted how the drug candidate would work against ATPase and ENTPDase targets using molecular methods. When hepatic injury was induced, there was a significant decrease in the levels of the GSH, CAT, and ENTPDase (p < 0.05) activities. In contrast, we found a noticeable rise in the MDA levels and ATPase activity. KAD-7 therapy resulted in lower levels of these activities overall (p < 0.05), as compared to the control levels. We found the compound to have a strong affinity for ATPase (-7.1 kcal/mol) and ENTPDase (-7.4 kcal/mol), and a better chemical reactivity than quercetin. It also met all drug-likeness parameters. Our study shows that KAD-7 can protect the liver from damage caused by FeSO4 by reducing oxidative stress and purinergic actions. Our studies indicate that KAD-7 could be developed as a therapeutic option since it can flexibly inhibit both ATPase and ENTPDase.

Differential Expression of Proteins Associated with Bipolar Disorder as Identified Using the PeptideShaker Software.

The prevalence of bipolar disorder (BD) in modern society is growing rapidly, but due to the lack of paraclinical criteria, its differential diagnosis with other mental disorders is somewhat challenging. In this regard, the relevance of proteomic studies is increasing due to the development of methods for processing large data arrays; this contributes to the discovery of protein patterns of pathological processes and the creation of new methods of diagnosis and treatment. It seems promising to search for proteins involved in the pathogenesis of BD in an easily accessible material-blood serum. Sera from BD patients and healthy individuals were purified via affinity chromatography to isolate 14 major proteins and separated using 1D SDS-PAGE. After trypsinolysis, the proteins in the samples were identified via HPLC/mass spectrometry. Mass spectrometric data were processed using the OMSSA and X!Tandem search algorithms using the UniProtKB database, and the results were analyzed using PeptideShaker. Differences in proteomes were assessed via an unlabeled NSAF-based analysis using a two-tailed Bonferroni-adjusted t-test. When comparing the blood serum proteomes of BD patients and healthy individuals, 10 proteins showed significant differences in NSAF values. Of these, four proteins were predominantly present in BD patients with the maximum NSAF value: 14-3-3 protein zeta/delta; ectonucleoside triphosphate diphosphohydrolase 7; transforming growth factor-beta-induced protein ig-h3; and B-cell CLL/lymphoma 9 protein. Further exploration of the role of these proteins in BD is warranted; conducting such studies will help develop new paraclinical criteria and discover new targets for BD drug therapy.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

EVALUATION OF COMPONENTS OF THE EXTRACELLULAR PURINERGIC SIGNALING SYSTEM IN HUMAN SEPSIS.

Objective: Extracellular purines such as adenosine triphosphate (ATP), uridine triphosphate (UTP), and uridine diphosphate (UDP) and the ATP degradation product adenosine are biologically active signaling molecules, which accumulate at sites of metabolic stress in sepsis. They have potent immunomodulatory effects by binding to and activating P1 or adenosine and P2 receptors on the surface of leukocytes. Here we assessed the levels of extracellular purines, their receptors, metabolic enzymes, and cellular transporters in leukocytes of septic patients. Methods: Peripheral blood mononuclear cells (PBMCs), neutrophils, and plasma were isolated from blood obtained from septic patients and healthy control subjects. Ribonucleic acid was isolated from cells, and mRNA levels for purinergic receptors, enzymes, and transporters were measured. Adenosine triphosphate, UTP, UDP, and adenosine levels were evaluated in plasma. Results: Adenosine triphosphate levels were lower in septic patients than in healthy individuals, and levels of the other purines were comparable between the two groups. Levels of P1 and P2 receptors did not differ between the two patient groups. mRNA levels of ectonucleoside triphosphate diphosphohydrolase (NTPDase) 1 or CD39 increased, whereas those of NTPDase2, 3, and 8 decreased in PBMCs of septic patients when compared with healthy controls. CD73 mRNA was lower in PBMCs of septic than in healthy individuals. Equilibrative nucleoside transporter (ENT) 1 mRNA concentrations were higher and ENT2, 3, and 4 mRNA concentrations were lower in PBMCs of septic subjects when compared with healthy subjects. Concentrative nucleoside transporter (CNT) 1 mRNA levels were higher in PBMCs of septic versus healthy subjects, whereas the mRNA levels of CNT2, 3, and 4 did not differ. We failed to detect differences in mRNA levels of purinergic receptors, enzymes, and transporters in neutrophils of septic versus healthy subjects. Conclusion: Because CD39 degrades ATP to adenosine monophosphate (AMP), the lower ATP levels in septic individuals may be the result of increased CD39 expression. This increased degradation of ATP did not lead to increased adenosine levels, which may be explained by the decreased expression of CD73, which converts AMP to adenosine. Altogether, our results demonstrate differential regulation of components of the purinergic system in PBMCs during human sepsis.

Phytochemical and biological characterization of aqueous extract of Vassobia breviflora on proliferation and viability of melanoma cells: involvement of purinergic pathway

ABSTRACT Vassobia breviflora belongs to the Solanaceae family, possessing biological activity against tumor cells and is a promising alternative for therapy. The aim of this investigation was to determine the phytochemical properties V. breviflora using ESI-ToF-MS. The cytotoxic effects of this extract were examined in B16-F10 melanoma cells and the relationship if any to purinergic signaling was involved. The antioxidant activity of total phenols, (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was analyzed, as well as production of reactive oxygen species (ROS) and nitric oxide (NO) was determined. Genotoxicity was assessed by DNA damage assay. Subsequently, the structural bioactive compounds were docked against purinoceptors P2X7 and P2Y1 receptors. The bioactive compounds found in V. breviflora were N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline, calystegine B, 12-O-benzoyl- tenacigenin A and bungoside B. In vitro cytotoxicity was demonstrated at concentration ranges of 0.1–10 mg/ml, and plasmid DNA breaks only at the concentration of 10 mg/ml. V. breviflora extracts affected hydrolysis by ectoenzymes, such as ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ectoadenosine deaminase (E-ADA) which control levels of degradation and formation of nucleosides and nucleotides. In the presence of substrates ATP, ADP, AMP and adenosine, the activities of E-NTPDase, 5´-NT or E-ADA were significantly modulated by V. breviflora. N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline presented higher binding affinity (according to receptor-ligand complex estimated binding affinity as evidenced by ∆G values) to bind to both P2X7 and P2Y1purinergic receptors. Our results suggest a putative interaction of V. breviflora bioactive compounds with growth inhibitory potential in B16-F10 melanoma and suggest that may be considered as promising compounds in melanoma and cancer treatment

Distribution of P2X3 purinoceptor-immunoreactive sensory nerve endings in the carotid body of Japanese macaque (Macaca fuscata).

In the carotid body of laboratory rodents, adenosine 5'-triphosphate (ATP)-mediated transmission is regarded as critical for transmission from chemoreceptor type I cells to P2X3 purinoceptor-expressing sensory nerve endings. The present study investigated the distribution of P2X3-immunoreactive sensory nerve endings in the carotid body of the adult male Japanese monkey (Macaca fuscata) using multilabeling immunofluorescence. Immunoreactivity for P2X3 was detected in nerve endings associated with chemoreceptor type I cells immunoreactive for synaptophysin. Spherical or flattened terminal parts of P2X3-immunoreactive nerve endings were in close apposition to the perinuclear cytoplasm of synaptophysin-immunoreactive type I cells. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), which hydrolyzes extracellular ATP, was localized in the cell body and cytoplasmic processes of S100B-immunoreactive cells. NTPDase2-immunoreactive cells surrounded P2X3-immunoreactive terminal parts and synaptophysin-immunoreactive type I cells, but did not intrude into attachment surfaces between terminal parts and type I cells. These results suggest ATP-mediated transmission between type I cells and sensory nerve endings in the carotid body of the Japanese monkey, as well as those of rodents.

Beet leaf (beta vulgaris L.) extract attenuates iron-induced testicular toxicity: Experimental and computational approach

The purpose of this study was to investigate the protective effect of Beta vulgaris leaf extract (BVLE) on Fe2+-induced oxidative testicular damage via experimental and computational models. Oxidative testicular damage was induced via incubation of testicular tissue supernatant with 0.1 mM FeSO4 for 30 min at 37 °C. Treatment was achieved by incubating the testicular tissues with BVLE under the same conditions. The catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) levels, acetylcholinesterase (AChE), sodium-potassium adenosine triphosphatase (Na+/K + ATPase), ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase), glucose-6-phosphatase (G6Pase), and fructose-1,6-bisphosphatase (F-1,6-BPase) were all measured in the tissues. We identified the bioactive compounds present using high-performance liquid chromatography (HPLC). Molecular docking and dynamic simulations were done on all identified compounds using a computational approach. The induction of testicular damage (p < 0.05) decreased the activities of GSH, SOD, CAT, and ENTPDase. In contrast, induction of testicular damage also resulted in a significant increase in MDA and NO levels and an increase in ATPase, G6Pase, and F-1,6-BPase activities. BVLE treatment (p < 0.05) reduced these levels and activities compared to control levels. An HPLC investigation revealed fifteen compounds in BVLE, with quercetin being the most abundant. The molecular docking and MDS analysis of the present study suggest that schaftoside may be an effective allosteric inhibitor of fructose 1,6-bisphosphatase based on the interacting residues and the subsequent effect on the dynamic loop conformation. These findings indicate that B. vulgaris can protect against Fe2+-induced testicular injury by suppressing oxidative stress, acetylcholinesterase, and purinergic activities while regulating carbohydrate dysmetabolism.

88-OR: Targeted Drug Delivery to Human ß-Cells Using Novel Nanocapsule Delivery Vehicle for T1D Treatment

In type 1 diabetes (T1D), autoreactive immune cells destroy insulin secreting β-cells in pancreatic islets. Islet transplantation shows promise in restoring glucose homeostasis without exogenous insulin; however, long-term graft survival remains a critical barrier to the success of this treatment. Therapies aimed to protect or proliferate endogenous β-cells have off target affects and require targeted delivery to improve efficacy. We have developed a nanocapsule (NC) delivery system to specifically target and deliver cargo to human β-cells. We hypothesize NCs will selectively be taken up by human β-cells and deliver therapeutic cargo. NCs were synthesized with a water in oil emulsion evaporation technique. NCs were coated via ionic interactions with ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) antibody, a human β-cell specific marker or Exendin-4 conjugated to hyaluronic acid (HA-Ex4). NC size, size distribution, charge, stability, and drug release profiles were determined. NCs loaded with pentamidine (PTM) or Cy5 were cultured for 24-168h with human iPSC-derived β-cells (sBC) or injected into the tail vein of NOD-SCID mice. Confocal microscopy confirmed delivery of cargo to β-cells and pancreatic islets, viability, and cytotoxicity. NCs were 271 ± 7nm in size with excellent size homogeneity (polydispersity index 0.06 ± 0.03, n=3). NCs displayed a sustained release of cargo for &amp;gt;45 days (n=5). In vitro, we observed no cytotoxicity of NCs up to 168h (n=3) and a significant increase in β-cell death with PTM loaded ENTPD3 coated NCs compared to controls at 24 (p&amp;lt;0.001, n=4) and 72h (p&amp;lt;0.01, n=4). sBCs treated with PTM loaded ENTPD3 coated NCs had more insulin+ dead cells (88 ± 4%) than controls (p&amp;lt;0.01, n=4). ENTPD3 or HA-Ex4 NCs were internalized into sBCs in vitro and pancreatic islets in vivo respectively. Our data shows long term stability and in vivo targeting of NCs to β-cells and supports use of our NCs for targeted delivery of therapeutics to human β-cells to treat T1D. Disclosure J.Collins: None. K.Holcomb: None. A.Ocampo: None. A.Shilleh: None. H.A.Russ: Advisory Panel; Sigilon Therapeutics, Inc., Consultant; Prellis, Minutia, Eli Lilly and Company. N.L.Farnsworth: None. Funding JDRF (1-INO-2022-1231-S-B); National Institutes of Health (P30-DK116073)

Human soluble CD39 displays substrate inhibition in a substrate-specific manner

CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD1) metabolizes extracellular ATP and ADP to AMP. AMP is subsequently metabolized by CD79 to adenosine. CD39 activity is therefore a key regulator of purinergic signalling in cancer, thrombosis, and autoimmune diseases. In this study we demonstrate that soluble, recombinant CD39 shows substrate inhibition with ADP or ATP as the substrate. Although CD39 activity initially increased with increasing substrate concentration, at high concentrations of ATP or ADP, CD39 activity was markedly reduced. Although the reaction product, AMP, inhibits CD39 activity, insufficient AMP was generated under our conditions to account for the substrate inhibition seen. In contrast, inhibition was not seen with UDP or UTP as substrates. 2-methylthio-ADP also showed no substrate inhibition, indicating the nucleotide base is an important determinant of substrate inhibition. Molecular dynamics simulations revealed that ADP can undergo conformational rearrangements within the CD39 active site that were not seen with UDP or 2-methylthio-ADP. Appreciating the existence of substrate inhibition of CD39 will help the interpretation of studies of CD39 activity, including investigations into drugs that modulate CD39 activity.

CD73: Friend or Foe in Lung Injury.

Ecto-5'-nucleotidase (CD73) plays a strategic role in calibrating the magnitude and chemical nature of purinergic signals that are delivered to immune cells. Its primary function is to convert extracellular ATP to adenosine in concert with ectonucleoside triphosphate diphosphohydrolase-1 (CD39) in normal tissues to limit an excessive immune response in many pathophysiological events, such as lung injury induced by a variety of contributing factors. Multiple lines of evidence suggest that the location of CD73, in proximity to adenosine receptor subtypes, indirectly determines its positive or negative effect in a variety of organs and tissues and that its action is affected by the transfer of nucleoside to subtype-specific adenosine receptors. Nonetheless, the bidirectional nature of CD73 as an emerging immune checkpoint in the pathogenesis of lung injury is still unknown. In this review, we explore the relationship between CD73 and the onset and progression of lung injury, highlighting the potential value of this molecule as a drug target for the treatment of pulmonary disease.

Synthesis and structure–activity relationships of ticlopidine derivatives and analogs as inhibitors of ectonucleotidase CD39

Ticlopidine is an antithrombotic prodrug of the thienotetrahydropyridine family. For platelet inhibition it has to undergo oxidative ring-opening by cytochrome P450 enzymes. The resulting thiol reacts with a cysteine residue of the purinergic P2Y12 receptor on thrombocytes resulting in covalent receptor blockade. Ticlopidine in its intact, not-metabolized form was previously shown to inhibit ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, also known as cluster of differentiation (CD) 39). CD39 catalyzes the extracellular hydrolysis of ATP via ADP to AMP, which is further hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine. CD39 inhibition has been proposed as a novel strategy to increase the extracellular concentration of antiproliferative ATP, while decreasing immunosuppressive and cancer-promoting adenosine levels. In the present study, we performed an extensive structure–activity relationship (SAR) analysis of ticlopidine derivatives and analogs as CD39 inhibitors followed by an in-depth characterization of selected compounds. Altogether 74 compounds were synthesized, 41 of which are new, not previously described in literature. Benzotetrahydropyridines, in which the metabolically labile thiophene is replaced by a benzene ring, were discovered as a new class of allosteric CD39 inhibitors.

Caffeic acid attenuates neuroinflammation and cognitive impairment in streptozotocin-induced diabetic rats: Pivotal role of the cholinergic and purinergic signaling pathways

The present study evaluated the effect of caffeic acid (CA) on behavioral learning and memory tasks in the diabetic state. We also evaluated the effect of this phenolic acid on the enzymatic activities of acetylcholinesterase, ecto-nucleoside triphosphate diphosphohydrolase, ecto-5-nucleotidase and adenosine deaminase as well as on the density of M1R, α7nAChR, P2×7R, A1R, A2AR, and inflammatory parameters in the cortex and hippocampus of diabetic rats. Diabetes was induced by a single intraperitoneal dose of streptozotocin (55 mg/kg). The animals were divided into six groups: control/vehicle; control/CA 10 and 50 mg/kg; diabetic/vehicle; diabetic/CA 10 and 50 mg/kg, treated by gavage. The results showed that CA improved learning and memory deficits in diabetic rats. Also, CA reversed the increase in acetylcholinesterase and adenosine deaminase activities and reduced ATP and ADP hydrolysis. Moreover, CA increased the density of M1R, α7nAChR, and A1R receptors and reversed the increase in P2×7R and A2AR density in both evaluated structures. In addition, CA treatment attenuated the increase in NLRP3, caspase 1, and interleukin 1β density in the diabetic state; moreover, it increased the density of interleukin-10 in the diabetic/CA 10 mg/kg group. The results indicated that CA treatment positively modified the activities of cholinergic and purinergic enzymes and the density of receptors, and improved the inflammatory parameters of diabetic animals. Thus, the outcomes suggest that this phenolic acid could improve the cognitive deficit linked to cholinergic and purinergic signaling in the diabetic state.

The ectonucleotidases CD39 and CD73 on T cells: The new pillar of hematological malignancy.

Hematological malignancy develops and applies various mechanisms to induce immune escape, in part through an immunosuppressive microenvironment. Adenosine is an immunosuppressive metabolite produced at high levels within the tumor microenvironment (TME). Adenosine signaling through the A2A receptor expressed on immune cells, such as T cells, potently dampens immune responses. Extracellular adenosine generated by ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) molecules is a newly recognized 'immune checkpoint mediator' and leads to the identification of immunosuppressive adenosine as an essential regulator in hematological malignancies. In this Review, we provide an overview of the detailed distribution and function of CD39 and CD73 ectoenzymes in the TME and the effects of CD39 and CD73 inhibition on preclinical hematological malignancy data, which provides insights into the potential clinical applications for immunotherapy.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Synthesis, biological evaluation, and molecular modeling studies of a new series of imidazothiazole or imidazooxazole derivatives as inhibitors of ectonucleoside triphosphate diphosphohydrolases (NTPDases)

Synthesis, biological evaluation, and molecular modeling studies of a new series of imidazothiazole or imidazooxazole derivatives as inhibitors of ectonucleoside triphosphate diphosphohydrolases (NTPDases)

Overexpression of TcNTPDase-1 Gene Increases Infectivity in Mice Infected with Trypanosoma cruzi.

Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).

Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-β1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor β1 (TGF-β1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-β1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-β1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-β1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-β1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-β1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-β1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Enzyme histochemistry: a useful tool for examining the spatial distribution of brain ectonucleotidases in (patho)physiological conditions.

Adenosine 5'-triphosphate (ATP) and other nucleotides and nucleosides, such as adenosine, are versatile signaling molecules involved in many physiological processes and pathological conditions in the nervous system, especially those with an inflammatory component. They can be released from nerve cells, glial cells, and vascular cells into the extracellular space where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and adenosine is regulated by cell-surface located enzymes ectonucleotidases that hydrolyze the nucleotide to the respective nucleoside. This review summarizes a histochemical approach for detection of ectonucleotidase activities in the cryo-sections of brain tissue. The enzyme histochemistry (EHC) might be used as suitable replacement for immunohistochemistry, since it gives information about both localization and activity, thus adding a functional component to a classical histological approach. With this technique, it is possible to visualize spatial distribution and cell-specific localization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and ecto-5'-nucleotidase (eN/CD73) activities during brain development, after different hormonal manipulations, during neurodegeneration, etc. EHC is also suitable for investigation of microglial morphology in different (patho)physiological conditions. Furthermore, the review describes how to quantify EHC results.

Immunohistochemical localization of vesicular nucleotide transporter in small intensely fluorescent (SIF) cells of the rat superior cervical ganglion

Our previous study reported that a part of small intensely fluorescent (SIF) cells in the rat superior cervical ganglion were innervated by P2X3 purinoceptor-expressing glossopharyngeal sensory nerve endings, suggesting the occurrence of adenosine 5′-triphosphate (ATP)-mediated transmission between them. The present study investigated the immunolocalization of vesicular nucleotide transporter (VNUT) in SIF cells of the superior cervical ganglion in male Wistar rats. VNUT was immunohistochemically localized in tyrosine hydroxylase-immunoreactive SIF cells and sympathetic postganglionic neurons, but not in a few SIF cells with immunoreactivity for dopamine beta-hydroxylase. P2X3-immunoreactive ramified nerve endings formed flat leaf-like or spherical terminal parts to surround some VNUT-immunoreactive SIF cells, but not other VNUT-immunoreactive SIF cells attached to ganglionic neurons. VNUT-immunoreactive SIF cells contained bassoon-immunoreactive products at the contacting surface of P2X3-immunoreactive nerve endings. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2, which hydrolyzes extracellular ATP, was observed in S100B-immunoreactive satellite glial cells surrounding VNUT-immunoreactive SIF cells, but not in the attachment surfaces between SIF cells and nerve endings with P2X3 immunoreactivity. The present results suggest that SIF cells release ATP by exocytosis to modulate the excitability of sensory nerve endings and postganglionic neurons in the superior cervical ganglion.