Ectonucleotidase Activity Research Articles

Platelets isolation and ectonucleotidase assay: Revealing functional aspects of the communication between the vasculature and the immune system

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5′-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.

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.

Ectonucleotidase Activity in Smooth Muscle Tissues of Rats with a Valproate Model of Autism.

Ectonucleotidases play an important role in regulating the level of extracellular nucleotides and nucleosides and are an important part of the regulation of the effects of adenosine and ATP on adenosine and P2 receptors, respectively. We have previously established the ambiguous effect of P2 receptor agonists on the contractile activity of smooth muscle tissue in rats with the valproate model of autism. In this work, HPLC was used to evaluate the activity of ectonucleotidases in the smooth muscle tissues of the internal organs of rats with a valproate model of autism. The activity of ectonucleotidases was significantly higher in the smooth muscle tissues of the duodenum, vas deferens, and bladder, but lower in the ileum and uterus. The results obtained make it possible to compare the activity of ectonucleotidases identified here with changes in P2 receptor-mediated contractility of smooth muscle tissues revealed in our previous experiments.

Abstract 5416: Purinergic P2Y6 receptor antagonists as potential anti tumor agents

Abstract Introduction: Cancer places a tremendous strain on society in both developed and economically less developed nations. It remains the most recurring and deadliest disease despite rapid advances in treatment modalities. The major contributors are the prevalence of known risk factors, urbanization, population growth and ageing. Cancer resurgence depends on the organ in which the tumor first appears, immune response, patient’s characteristics, disease stage and tumor microenvironment (TME). Among the several inflammatory molecules found within the TMEs, the levels of adenosine triphosphate (ATP) has been found to be high. In healthy tissue, the concentration of ATP in the extracellular space (eATP) is negligible and does not accumulate significantly due to the activity of ectonucleotidases. However, tissue injury, cellular stress and chemotherapy mediated cell death can increase the levels of ATP in the TME. The eATP exerts its effect mainly through purinergic P2 receptors expressed by most malignant cells. We have previously shown that eATP promoted migration and invasion of cancer cells in vitro through increased expression of the enzyme cyclooxygenase 2 (COX-2) (Sharma et al., 2021). We showed that eATP mediated COX-2 expression is through P2 receptors (Akter et al., 2021). Accordingly, we hypothesized that blocking the P2 receptors would abolish the COX-2-mediated migration of tumor cells and tested the same in an animal model. Methodology: Tumors were generated in 8-15-week-old male C57BL/6 mice (20-25 g weight) through subcutaneous injection of syngeneic EL4 lymphoma cells in the left flank of the mice. When tumors were palpable around day 10, saline or P2 receptor, antagonists were directly injected into the center of the tumor and mice were divided into three groups: saline (vehicle control), suramin (broad-spectrum P2 receptor antagonist), and MRS 2578 (P2Y6 receptor-specific antagonist). Drugs were chosen based on prior in vitro research and a vernier caliper was used to assess the growth of the tumor every day. Behavioral tests were performed on day 8 (prior to P2 receptor injections) and days 11, 14 and 17 post-tumor injection. Mice were continuously monitored for their health and were sacrificed on day 18 with deep CO2 anesthesia and cervical dislocation. Results: We found that suramin-injected tumors had a substantial reduction in tumor volume when compared to saline-injected ones. Intra-tumoral injections of P2 receptor antagonists such as MRS-2578 also resulted in similar outcomes as suramin. In tumors treated with P2 receptor antagonists, the expression of COX-2, cell cycle proteins and epithelial-to-mesenchymal (EMT) markers, which aid in metastasis, were significantly downregulated, indicating a suppressive effect. Therefore, we propose that P2 receptor-dependent anti-inflammatory drugs (PBAIDs) could be considered a potential alternative therapeutic option for preventing cancer recurrence. Citation Format: Khagendra Ghimeray, Shilpa Sharma, Ravi Shankar Akundi. Purinergic P2Y6 receptor antagonists as potential anti tumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5416.

Activity of enzymes destroying extracellular nucleotides in the tissues of rats with the valproate model of autism

BACKGROUND: Ectonucleotidases hydrolyze extracellular nucleotides and thus can control the effect of these substances on purinergic P1 and P2 receptors.
 AIM: To evaluate the activity of ectonucleotidases in the smooth muscle tissues of internal organs of 9-month-old rats with the valproate model of autism using high-performance liquid chromatography.
 MATERIAL AND METHODS: Autism was modeled in outbred Wistar rats by administering valproic acid (500 mg/kg) subcutaneously to pregnant females on days 12–13 of pregnancy. The born offspring were used in the study when the rats reached 270±8 days. Animals were guillotined under light ether anesthesia, the bladder, uterus, vas deferens, and duodenum were isolated, and smooth muscle tissue samples were prepared. Total ectonucleotidase activity was determined by incubating tissue samples with adenosine triphosphate (reaction substrate) for 10 minutes with further assessment of the content of the substrate and reaction products (adenosine diphosphate, adenosine monophosphate) in the incubate using high-performance liquid chromatography. Mathematical and statistical processing of the results was carried out using Microsoft Excel and IBM SPSS Statistics 26.0 software. Group comparisons were made using the nonparametric Mann–Whitney U test. Differences were considered significant at p 0.05.
 RESULTS: In rats with the valproate model of autism, the activity of ectonucleotidases in the smooth muscle tissues of the vas deferens (609.5±153.9) and uterus (232.7±2) was significantly lower than control values (2114.6±524.3, p=0.040; 539.6±63.5, p=0.010, respectively). In the duodenum (1808.4±184.5) and bladder (1021.3±280.7) we did not find a significant difference compared to the control values (2115.0±393.3, p=0.712; 2302.3±615.8, p=0.274, respectively). This study allows us to evaluate the possible contribution of purinergic transmission to the changes we found earlier in the contractile activity of smooth muscle tissue in rats with the valproate model of autism.
 CONCLUSION: In 9-month-old rats with a model of autism, the activity of ectonucleotidases in the smooth muscle tissues of the reproductive organs is reduced; no such changes were found in the tissues of the intestines and bladder.

Low ectonucleotidase activity and increased neutrophil-platelet aggregates in patients with antiphospholipid syndrome

AbstractMany patients with antiphospholipid syndrome had decreased ectonucleotidase activity on neutrophils and platelets, which enabled extracellular nucleotides to trigger neutrophil-platelet aggregates. This phenotype was replicated by treating healthy neutrophils and platelets with patient-derived antiphospholipid antibodies or ectonucleotidase inhibitors.

Caffeine reduces viability, induces apoptosis, inhibits migration and modulates the CD39/CD73 axis in metastatic cutaneous melanoma cells.

We aimed to evaluate the effect of caffeine on viability, apoptosis, migration, redox profile and modulatory effect of the purinergic system of cutaneous melanoma cells. The melanoma cells SK-MEL-28 and non-tumoural CCD-1059sk cells were treated for 24 h with different concentrations of caffeine. Cell viability was evaluated by a biochemical assay and fluorescence microscopy, and flow cytometry assessed apoptosis induction. A wound-healing assay assessed cell migration. The redox profile was evaluated by the levels of markers of reactive oxygen species (ROS), nitric oxide (NOx), total thiols (PSH) and non-protein thiols (NPSH). RT-qPCR and flow cytometry assessed the expression of CD39 and CD73. ATPase/ADPase and AMPase enzyme activities were evaluated by hydrolysis of ATP, ADP and AMP nucleotides. A bioluminescent assay assessed extracellular ATP levels. Caffeine significantly reduced melanoma cell viability and migration and did not affect non-tumoural cells. Caffeine increased ROS levels and improved PSH levels in melanoma cells. Furthermore, caffeine reduced CD39 and CD73 expression, decreased ATP, ADP and AMP nucleotide hydrolysis and increased extracellular ATP levels. We have shown that caffeine reduces metastatic cutaneous melanoma cell viability and migration, induces ROS generation and improves PSH levels. In an unprecedented manner, we also showed that caffeine reduces the expression of CD39 and CD73 and, consequently, ATPase/ADPase/AMPase hydrolytic activity of ectonucleotidases, thus displacing the CD39/CD73 axis and increasing extracellular ATP levels. Therefore, caffeine may be an interesting compound for clinical trials with the CD39/CD73 axis as a therapeutic target.

Abstract P3113: Apyrase But Not Dipyridamole Improves Cardiac Function In Ovariectomized High-fat Diet Mice

Objectives: Postmenopausal women with metabolic disease are predisposed to develop cardiovascular disease. Our previous experiments have shown that these women experience purinergic/adenosinergic signaling alteration resulting in diastolic dysfunction. We hypothesize that these signaling alterations are reversible and to demonstrate we used exogenous CD39 agonist (apyrase) and an ENT-1 inhibitor (dipyridamole), in a murine model simulating postmenopausal women. Methods: We fed 23 C57BL/6 female mice a HFD (40% kcal) from 5 weeks of age and performed ovariectomy at 12 weeks. Intraperitoneal (IP) injections were given daily to three groups for 10 days at 28 weeks. The following groups and dosages were used: apyrase (9 mice, 200U/kg), dipyridamole (9 mice, 20mg/kg), and saline (5 mice). At 30 weeks, mice were assessed with an EchoMRI for body composition, and transthoracic echocardiography (TTE) used for evaluation of cardiac function while mice were anesthetized with 2.5% isoflurane. Results: Our apyrase-treated mice, had significantly lowered values in E/E’, E/A, with increased Ejection Fraction and Fraction Shortening when compared to the saline treated groups, analyzed with t-test. Apyrase also had lower body weight, lean mass, fat mass, and heart/body weight percentage, when compared to saline (p<0.05). Dipyridamole had significant increase in LV diastolic volume and IVRT when compared to our saline group. Conclusion: Our findings could suggest that increased ectonucleotidase activity may ameliorate diastolic dysfunction experienced in our murine model. Future studies are needed to confirm its effect in postmenopausal women.

Circulating Ectonucleotidases Signal Impaired Myocardial Perfusion at Rest and Stress.

Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants (P=0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P<0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P<0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.

Neuromodulatory effect of the combination of metformin and vitamin D3 triggered by purinergic signaling in type 1 diabetes induced-rats

Several studies have indicated the vitamin D deficiency in the development of macro- and microvascular complications of diabetes mellitus (DM) including DM-related cognitive dysfunction. The purinergic system plays an important role in the modulation of a variety of mechanisms, including neuroinflammation, plasticity, and cell-cell communication. In addition, purines, their receptors, and enzymes can regulate the purinergic axis at different levels in type 1 DM (T1DM). This study evaluated the effects of vitamin D3 alone or in combination with metformin in the behavioral performance of streptozotocin-induced T1DM rats. The effects of this combination on the metabolism of ATP and ADP were also studied by NTPDase (CD39), AMP by 5′-nucleotidase (CD73), and adenosine by adenosine deaminase (E-ADA) in the brain and peripheral lymphocytes of type 1 diabetic STZ-induced rats. The results showed that anxiety and memory loss from the DM condition reverted after 30 days of vitamin D3 treatment. Furthermore, the DM state affected systemic enzymes, with no effect on the central enzymes hydrolyzing extracellular nucleotides and nucleosides. Vitamin D3 treatment positively regulated ectonucleotidase (NTPDase and 5′-nucleotidase) activity, E-ADA, and the purinergic receptors as a mechanism to prevent oxidative damage in the cerebral cortex of T1DM rats. A neuroprotector effect of vitamin D3 through adenosine signaling was also observed, by regulating A1 and A2A receptors proteins levels. The present findings suggest that purinergic signaling through vitamin D3 modulation may be a novel alternative strategy for T1DM treatment, and may compensate for the negative changes in the central nervous system.

Salt-loading promotes extracellular ATP release mediated by glial cells in the hypothalamic paraventricular nucleus of rats

Previously, we have shown that purinergic signalling is involved in the control of hyperosmotic-induced sympathoexcitation at the level of the PVN, via activation of P2X receptors. However, the source(s) of ATP that drives osmotically-induced increases in sympathetic outflow remained undetermined. Here, we tested the two competing hypotheses that either (1) higher extracellular ATP in PVN during salt loading (SL) is a result of a failure of ectonucleotidases to metabolize ATP; and/or (2) SL can stimulate PVN astrocytes to release ATP. Rats were salt loaded with a 2 % NaCl solution replacing drinking water up to 4 days, an experimental model known to cause a gradual increase in blood pressure and plasma osmolarity. Immunohistochemical assessment of glial-fibrillary acidic protein (GFAP) revealed increased glial cell reactivity in the PVN of rats after 4 days of high salt exposure. ATP and adenosine release measurements via biosensors in hypothalamic slices showed that baseline ATP release was increased 17-fold in the PVN while adenosine remained unchanged. Disruption of Ca2+-dependent vesicular release mechanisms in PVN astrocytes by virally-driven expression of a dominant-negative SNARE protein decreased the release of ATP. The activity of ectonucleotidases quantified in vitro by production of adenosine from ATP was increased in SL group. Our results showed that SL stimulates the release of ATP in the PVN, at least in part, from glial cells by a vesicle-mediated route and likely contributes to the neural control of circulation during osmotic challenges.

Abstract 15260: Role Of Purinergic Dysfunction in Worsened Outcomes for Females Undergoing Cardiac Surgery

Objective: To determine whether purinergic signaling plays a role in the adverse outcome in females presenting for cardiac surgery Methods: Right atrial tissue was harvested from 80 patients undergoing CABG surgery before and after and cardiopulmonary bypass (CBP). Human tissue experimentation was complemented by experiments in mice subject to a high fat diet and ovariectomy. Western blot and immunohistochemistry was carried out to interrogate the molecular derangements. Results: Females (n=40) had increased diastolic dysfunction (56% vs. 31%, p = .034) and length of stay (7.9 vs. 5.4 days, p = .02). We found that CD39, responsible for the enzymatic lysis of inflammatory extracellular ATP, was significantly decreased in women after CBP. Moreover, female patients demonstrated increased expression of ADORA2B, which acts as a pro-fibrotic agent responsive to adenosine as well as increased CD73, which is responsible for the lysis of adenosine monophosphate into adenosine. Finally, females demonstrated decreased adenosine deaminase, which breaks adenosine into inosine. Female patients had increased fibrosis and decreased angiogenesis (Figure 1a). Mouse models showed a similar decrease in purinergic ectonucleotidase activity as a result of ovariectomy and estrogen loss when compared to controls (Figure 1b). Conclusions: Purinergic signaling may serve as a key upstream mechanism responsible for the molecular derangements which underlie poorer outcomes for women following CABG surgery.

CD69 expression on regulatory T cells protects from immune damage after myocardial infarction.

Increasing evidence has pointed to the important function of T cells in controlling immune homeostasis and pathogenesis after myocardial infarction (MI), although the underlying molecular mechanisms remain elusive. In this study, a broad analysis of immune markers in 283 patients revealed significant CD69 overexpression on Tregs after MI. Our results in mice showed that CD69 expression on Tregs increased survival after left anterior descending (LAD) coronary artery ligation. Cd69-/- mice developed strong IL-17+ γδT cell responses after ischemia that increased myocardial inflammation and, consequently, worsened cardiac function. CD69+ Tregs, by induction of AhR-dependent CD39 ectonucleotidase activity, induced apoptosis and decreased IL-17A production in γδT cells. Adoptive transfer of CD69+ Tregs into Cd69-/- mice after LAD ligation reduced IL-17+ γδT cell recruitment, thus increasing survival. Consistently, clinical data from 2 independent cohorts of patients indicated that increased CD69 expression in peripheral blood cells after acute MI was associated with a lower risk of rehospitalization for heart failure (HF) after 2.5 years of follow-up. This result remained significant after adjustment for age, sex, and traditional cardiac damage biomarkers. Our data highlight CD69 expression on Tregs as a potential prognostic factor and a therapeutic option to prevent HF after MI.

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.

Postnatal Development of the Purinergic Signaling System in the preBötzinger Complex; Implications for the Hypoxic Ventilatory Response

The hypoxic ventilatory response (HVR) comprises a carotid body‐mediated increase in ventilation followed by a secondary, CNS‐mediated decrease, the hypoxic respiratory depression (HRD), that is larger and potentially life‐threatening in newborn mammals. The mechanisms responsible for the greater HRD in newborns are unknown, but purinergic signaling via P2 and P1 receptor (R) mechanisms helps shape this 2° phase. Shortly after the 1° increase in ventilation, astrocytes release ATP in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation), which stimulates breathing via P2Y1Rs and attenuates the HRD. Extracellular ATP (ATPe) is degraded to adenosine (ADOe), which inhibits breathing and is implicated in the HRD. Thus, the impact of ATP on the HVR is determined by the balance between the actions of ATP and ADOe, that could change developmentally and contribute to the greater HRD of newborns. Our goal was to characterize how the many factors that determine the balance between ATP and ADOe signaling change during development, including: P2Y1Rs; ADO A1Rs; ectonucleotidases (ECTOs) that degrade ATPe to ADOe; ADOe clearance by equilibrative nucleoside transporters (ENTs) via transmembrane transport of ADO down its concentration gradient; and ADO kinase (ADK), which is not mature in the cortex until postnatal day (P) 14, but facilitates ADOe clearance by converting ADOi into AMP so that ADOi&lt;ADOe. We used rhythmic preBötC slice preparations to characterize developmental changes in: i) the effects of P2Y1R and ADOR signaling on preBötC rhythm; ii) ECTO activity; and iii) the impact of ENTs and ADK on baseline preBötC rhythm and preBötC responses to ATP and ADO that were locally injected to simulate hypoxia. iv) In vivo head‐out plethysmography was used to assess the role of ADOe clearance mechanisms in shaping the developing HVR.i) The frequency (freq) increase evoked by ATP in the preBötC doubled between P0‐6 and P9‐12, but the P2Y1R agonist MRS 2365‐evoked increase did not change. The A1R inhibition of freq was of similar magnitude but greater duration at P0‐6 compared to P9‐12. ii) ECTO activity, assessed by incubating preBötC tissue in ATP and measuring PO4, was greater at P9‐12. iii) The ADO freq inhibition was 2‐fold longer in ENT knockout mice compared to WT mice at all ages. ADK inhibition (ABT 702) inhibited baseline freq only at P9‐12. iv) Knockout of ENTs blunted the freq response to hypoxia (10% O2) in vivo, but increased the VT response at all ages. Introduction of constitutively active ADK into the brain produced P0‐2 ADKtg mice that responded to hypoxia with an adult‐like, sustained increase in VE/VO2; P0‐2 WT C57BL6 did not respond to hypoxia. This difference disappeared by P6‐8. Surprisingly, the mature HVR of P0‐2 ADKtg mice reflected a greater hypoxic depression of VO2, not a greater increase in VE.Data suggest preBötC sensitivity to P2Y1 and A1Rs does not change, while ECTO activity increases developmentally. ADO actions are prolonged in P0‐6 mice, suggesting that ADOe clearance mechanisms mature postnatally. ENTs appear to play a similar role from birth, which may involve clearing ADOe from the preBötC during hypoxia. ADK maturation also appears important in the development of an adaptive HVR. However, the role of ADK in modulating VE vs metabolic responses to hypoxia remains to be elucidated.

High levels of extracellular ATP lead to different inflammatory responses in COVID-19 patients according to the severity.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly impacted the world and has driven many researchers into the pathophysiology of COVID-19. In the findings, there is a close association between purinergic signaling and the immune response. Then, this study aimed to evaluate alterations in the purinergic signaling in COVID-19 patients according to range severity. We divided the COVID-19 patients into moderate and severe cases following the guideless of NIH and WHO, together with clinical characteristics. The blood samples were collected to obtain PBMCs and platelets. We analyzed the ectonucleotidase activities through ATP, ADP, AMP, Ado hydrolysis, E-NTPDase1 (CD39), and 5′-NT (CD73) expression by flow cytometry in total leukocytes. The extracellular ATP was measured by bioluminescence, and cytokines were analyzed by flow cytometry. We observed a decrease in ATP hydrolysis and increased AMP hydrolysis in PBMCs for both groups. In severe cases, ATP hydrolysis was raised for the platelets, while ADP and AMP hydrolysis have risen significantly in both groups. Additionally, there was a significant increase in ADP hydrolysis in severe cases compared to moderate cases. In addition, we observed an increase in the ADA activity in platelets of moderate patients. Moderate and severe cases showed increased expression of CD39 and CD73 in total leukocytes. To finalize the purinergic signaling, extracellular ATP was increased in both groups. Furthermore, there was an increase in IL-2, IL-6, IL-10, and IL-17 in moderate and severe groups. Thus, for the first time, our findings confirm the changes in purinergic signaling and immune response in COVID-19, in addition to making it more evident that the severity range directly impacts these changes. Therefore, the therapeutic potential of the purinergic system must be highlighted and studied as a possible target for the treatment of SARS-CoV-2 disease.Key messages COVID-19 patients exhibit alterations in purinergic system and immune response.High levels of extracellular ATP lead to different inflammatory responses.CD39 and CD73 expression were increased in COVID-19 patients.Cytokines IL-2, IL-6, IL-10, and IL-17 also were altered in these patients.The purinergic system may be a possibility target to SARS-CoV-2 treatments. Graphical abstract

Tannic Acid Attenuates Peripheral and Brain Changes in a Preclinical Rat Model of Glioblastoma by Modulating Oxidative Stress and Purinergic Signaling.

Glioblastoma (GB) is a highly aggressive and invasive brain tumor; its treatment remains palliative. Tannic acid (TA) is a polyphenol widely found in foods and possesses antitumor and neuroprotective activities. This study aimed to investigate the effect of TA on oxidative stress parameters and the activity of ectonucleotidases in the serum, platelets, and lymphocytes and/or in the brain of rats with preclinical GB. Rats with GB were treated intragastrically with TA (50mg/kg/day) for 15days or with a vehicle. In the platelets of the animals with glioma, the adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolysis and the catalase (CAT) activity decreased. Besides, the adenosine diphosphate (ADP) hydrolysis, adenosine (Ado) deamination, and the reactive oxygen species (ROS) and nitrite levels were increased in glioma animals; however, TA reversed ROS and nitrite levels and AMP hydrolysis alterations. In lymphocytes from animals with glioma, the ATP and ADP hydrolysis, as well as Ado deamination were increased; TA treatment countered this increase. In the brain of the animals with glioma, the ROS, nitrite, and thiobarbituric acid reactive substance (TBARS) levels increased and the thiol (SH) levels and CAT and superoxide dismutase (SOD) activities were decreased; TA treatment decreased the ROS and TBARS levels and restored the SOD activity. In the serum of the animals with glioma, the ATP hydrolysis decreased; TA treatment restored this parameter. Additionally, the ROS levels increased and the SH and SOD activity decreasedby glioma implant; TA treatment enhanced nitrite levels andreversed SOD activity. Altogether, our results suggest that TA is an important target in the treatment of GB, as it modulates purinergic and redox systems.

The Ecto-5'nucleotidase/CD73 Mediates Leishmania amazonensis Survival in Macrophages.

Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, β-methyleneadenosine 5′-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.

Effect of Thiazolidin-4-one Against Lipopolysaccharide-Induced Oxidative Damage, and Alterations in Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Cultured Astrocytes.

Astrocytes play multiple important roles in brain physiology. However, depending on the stimuli, astrocytes may exacerbate inflammatory reactions, contributing to the development and progression of neurological diseases. Therefore, therapies targeting astrocytes represent a promising area for the development of new brain drugs. Thiazolidinones are heterocyclic compounds that have a sulfur and nitrogen atom and a carbonyl group in the ring and represent a class of compounds of great scientific interest due to their pharmacological properties. The aim of this study was to investigate the effect of 3-(3-(diethylamino)propyl)-2-(4-(methylthio)phenyl)thiazolidin-4-one (DS27) on cell proliferation and morphology, oxidative stress parameters, activity of the enzymes ectonucleotidases and acetylcholinesterase (AChE) and interleukin 6 (IL-6) levels in primary astrocyte cultures treated with lipopolysaccharide (LPS), to model neuroinflammation. The astrocyte culture was exposed to LPS (10μg/ml) for 3h and subsequently treated with compound DS27 for 24 and 48h (concentrations ranging to 10-100μM). LPS induced an increase in astrocyte proliferation, AChE activity, IL-6 levels, oxidative damage, ATP and ADP and a reduction in AMP hydrolysis in rat primary astrocyte cultures. DS27 treatment was effective in reversing these alterations induced by LPS. Our findings demonstrated that DS27 is able to modulate cholinergic and purinergic signaling, redox status, and the levels of pro-inflammatory cytokines in LPS-induced astrocyte damage. These glioprotective effects of DS27 may be very important for improving neuroinflammation, which is associated with many brain diseases.