Plasma Adenosine Research Articles

Hyperoxia Improves Hemodynamic Status During Head-up Tilt Testing in Healthy Volunteers: A Randomized Study.

Head-up tilt test is useful for exploring neurally mediated syncope. Adenosine is an ATP derivative implicated in cardiovascular disturbances that occur during head-up tilt test. The aim of the present study was to investigate the impact of hyperoxia on adenosine plasma level and on hemodynamic changes induced by head-up tilt testing.Seventeen healthy male volunteers (mean age 35 ± 11 years) were included in the study. The experiment consisted of 2 head-up tilt tests, 1 session with subjects breathing, through a mask, medical air (FiO2 = 21%) and 1 session with administration of pure oxygen (FiO2 = 100%) in double-blind manner. Investigations included continuous monitoring of hemodynamic data and measurement of plasma adenosine levels.No presyncope or syncope was found in 15 of the 17 volunteers. In these subjects, a slight decrease in systolic blood pressure was recorded during orthostatic stress performed under medical air exposure. In contrast, hyperoxia led to increased systolic blood pressure during orthostatic stress when compared with medical air. Furthermore, mean adenosine plasma levels decreased during hyperoxic exposure before (0.31 ± 0.08 μM) and during head-up tilt test (0.33 ± 0.09 μM) when compared with baseline (0.6 ± 0.1 μM). Adenosine plasma level was unchanged during medical air exposure at rest (0.6 ± 0.1 μM), and slightly decreased during orthostatic stress. In 2 volunteers, the head-up tilt test induced a loss of consciousness when breathing air. In these subjects, adenosine plasma level increased during orthostatic stress. In contrast, during hyperoxic exposure, the head-up tilt test did not induce presyncope or syncope. In these 2 volunteers, biological study demonstrated a decrease in adenosine plasma level at both baseline and during orthostatic stress for hyperoxic exposure compared with medical air.These results suggest that hyperoxia was able to increase blood pressure during head-up tilt test via a decrease in plasma adenosine concentration. Our results also suggest that adenosine receptor antagonists are worth trying in neurocardiogenic syncope.

Idiopathic Paroxysmal Atrio-Ventricular Block. What is The Mechanism?

Idiopathic paroxysmal atrioventricular (AV) block poses a true diagnostic challenge. What is clear about this entity is the confusion about its definition and consequently about its etiology. According to certain sources, the diagnosis of this block requires the lack of a structural cardiac pathology that justifies the observed manifestations and an absence of electrocardiographic disorders prior to an episode. The clinical presentation of idiopathic paroxysmal AV block does not differ from that of another cardiogenic syncope or of a vasovagal syncope with a significant cardioinhibitory component. With respect to the mechanism that explains this block, it has been postulated that patients with low basal adenosine levels exhibit hyperaffinity of the A2 receptors of the AV node. Variations in plasma adenosine levels may favor episodes of paroxysmal AV block. The diagnosis of this block is complex and can require years to determine. Routine electrophysiological examination of these patients is not cost effective due to the low sensitivity and specificity of this approach. Numerous groups have supported the use of an implantable loop recorder to substantiate AV block paroxysms and assess their clinical correlations. Permanent stimulation devices are utilized to reduce syncopal recurrence.

Biomarker sensitivity of plasma adenosine levels to predict heart failure in dogs

Biomarker sensitivity of plasma adenosine levels to predict heart failure in dogs

Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus)

In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity

AbstractErythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

The clinical significance of serum neopterin and adenosine deaminase in patients with secondary hemophagocytic lymphohistiocytosis

To investigate the significance of plasma neopterin (Npt) and adenosine deaminase (ADA) in patients with secondary hemophagocytic lymphohistiocytosis (sHLH). Serum specimens from 39 patients with newly diagnosed sHLH, 10 sHLH patients who had achieved clinical remission after treatment, and 15 healthy controls were collected.Serum Npt level was detected by enzyme linked immunosorbent assay (ELISA) and ADA activity was tested by kinetic method. Npt and ADA values in sHLH group were significantly higher than those in control group [Npt: (164.6 ± 90.0) nmol/L vs (7.9 ± 3.6) nmol/L;ADA: (117.2 ± 70.2) U/L vs (11.6 ± 4.0) U/L;all P < 0.001]. Among the 10 sHLH patients who obtained effective clinical treatment, posttreatment levels of Npt and ADA were significantly lower than pretreatment data [Npt: (17.5 ± 10.9) nmol/L vs (170.6 ± 117.9) nmol/L;ADA: (22.5 ± 15.5) U/L vs (98.8 ± 52.6) U/L;all P < 0.05]. The Npt level in sHLH patients was positively correlated with the levels of serum soluble interleukin-2 receptor (sCD25) and serum ferritin (r = 0.526 and r = 0.507) ; while ADA activity had linear relationship with the level of lactate dehydrogenase (r = 0.646) .Receiver operating characteristic (ROC) curve analysis showed that 148.1 nmol/L was the critical value of serum Npt for the diagnosis of lymphoma associated hemophagocytic syndrome (LAHS) and the sensitivity and specificity were 70.0% and 78.9%, respectively. As to ADA, 103.1 U/L was the critical value for the diagnosis of LAHS and the sensitivity and specificity were 75.0% and 84.2%, respectively. The sensitivity and specificity of combined parameters of Npt and ADA were 90.0% and 94.7%, respectively. It is concluded that Npt and ADA have great importance in the diagnosis and evaluation of therapeutic effect in patients with sHLH.Npt and ADA provide potential evidence to diagnose patients who are suspected with LAHS.

Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Prevents Hypoxia-Induced Tissue Injury in High Altitude By Inducing 2,3-Bisphosphoglycerate

To function effectively in O2 uptake, delivery and release, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by allosteric modulators, especially 2,3-bisphophosphoglycerate (2,3-BPG). Earlier studies had shown that elevated erythrocyte 2,3-BPG level is correlated to increased availability of oxygen to tissues in high altitudes. However, nothing is known how 2,3-BPG is induced in high altitude. Recent study revealed that plasma adenosine is increased in patient and mice with sickle cell disease and elevated adenosine signaling via A2B receptor (ADORA2B) induces 2,3-BPG and thereby promotes deoxy-sickle hemoglobin and subsequent sickling. However, whether adenosine is induced and its effect in 2,3-BPG induction in high altitude are unknown. To address this question, we recruited 24 low-land volunteers and placed them in high altitude for different time points. Here we report that: 1) plasma adenosine, a molecule well known to be induced under hypoxia, is increased and its elevation is correlated to elevated erythrocyte 2,3-BPG levels and decreased Hb-O2 affinity; and 2) elevated plasma adenosine is correlated to increased circulating ecto-5’-nucleotidase (CD73), a key enzyme responsible for generation of extracellular adenosine. Similar to humans, increased circulating CD73 activity, plasma adenosine levels and erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were observed in wild type (WT) mice under hypoxia condition mimicking our human high altitude studies. Moreover, we found that hypoxia-induced increased plasma adenosine, erythrocyte 2,3-BPG and decreased Hb-O2 binding affinity were significantly attenuated in CD73-deficient mice (CD73-/-). As such, hypoxia-induced tissue injury and cell apoptosis were significantly elevated in CD73-/- mice compared to WT mice. Mechanistically, we provide both in vitro and in vivo genetic evidence that erythrocyte adenosine A2B receptor (ADORA2B) is essential for 2,3-BPG induction and subsequent O2 release. This finding led us to further discover that AMP-Activated Protein Kinase (AMPK) functions downstream of ADORA2B underlying adenosine-induced 2,3-BPG induction and O2 release by direct phosphorylation of 2,3-BPG mutase, a key enzyme for 2,3-BPG production. Finally, we demonstrated that treatment of metformin, a FDA approved potent AMPK agonist, induced erythrocyte 2,3-BPG levels and triggered O2 release and thereby prevented hypoxia-induced tissue injury in both CD73-/- mice and erythrocyte specific ADORA2B-deficient mice. Overall, our studies have revealed that erythrocyte ADORA2B-mediated AMPK activation is a novel mechanism underlying hypoxia-induced 2,3-BPG levels and thereby highlight its beneficial role to prevent hypoxia-induced tissue injury. DisclosuresNo relevant conflicts of interest to declare.

Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity

Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. DisclosuresNo relevant conflicts of interest to declare.

Regulation of the skeletal muscle blood flow in humans.

In humans, skeletal muscle blood flow is regulated by an interaction between several locally formed vasodilators, including NO and prostaglandins. In plasma, ATP is a potent vasodilator that stimulates the formation of NO and prostaglandins and, very importantly, can offset local sympathetic vasoconstriction. Adenosine triphosphate is released into plasma from erythrocytes and endothelial cells, and the plasma concentration increases in both the feed artery and the vein draining the contracting skeletal muscle. Adenosine also stimulates the formation of NO and prostaglandins, but the plasma adenosine concentration does not increase during exercise. In the skeletal muscle interstitium, there is a marked increase in the concentration of ATP and adenosine, and this increase is tightly coupled to the increase in blood flow. The sources of interstitial ATP and adenosine are thought to be skeletal muscle cells and endothelial cells. In the interstitium, both ATP and adenosine stimulate the formation of NO and prostaglandins, but ATP has also been suggested to induce vasoconstriction and stimulate afferent nerves that signal to increase sympathetic nerve activity. Adenosine has been shown to contribute to exercise hyperaemia, whereas the role of ATP remains uncertain due to lack of specific purinergic receptor blockers for human use. The purpose of this review is to address the interaction between vasodilator systems and to discuss the multiple proposed roles of ATP in human skeletal muscle blood flow regulation.

Plasma ATP is Required for Neutrophil Activation in a Mouse Sepsis Model

Our previous work has shown that polymorphonuclear neutrophils (PMNs) require cellular adenosine triphosphate (ATP) release and autocrine purinergic signaling for their activation. Here we studied in a mouse model of cecal ligation and puncture (CLP) whether sepsis affects this purinergic signaling process and thereby alters PMN responses after sepsis. Using high-performance liquid chromatography, we found that plasma ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations increased up to 6-fold during the first 8 h after CLP, reaching top levels that were significantly higher than those in sham control animals without CLP. Although leukocyte and PMN counts in sham animals increased significantly after 4 h, these blood cell counts decreased in sepsis animals. CD11b expression on the cell surface of PMNs of septic animals was significantly higher compared with sham and untreated control animals. These findings suggest increased PMN activation and sequestration of PMN from the circulation after sepsis. Plasma ATP levels correlated with CD11b expression, suggesting that increased ATP concentrations in plasma contribute to PMN activation. We found that treatment of septic mice with the ATP receptor antagonist suramin diminished CD11b expression, indicating that plasma ATP contributes to PMN activation by stimulating P2 receptors of PMNs. Increased PMN activation can protect the host from invading microorganisms. However, increased PMN activation can also be detrimental by promoting secondary organ damage. We conclude that pharmacological targeting of P2 receptors may allow modulation of PMN responses in sepsis.

Adenosine signaling via A2B receptor regulates erythrocyte 2,3‐bisphosphate induction at high altitude (885.6)

To function effectively in O2 uptake, and unload, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)‐O2 affinity by allosteric modulators. One of the best known allosteric modulators is 2,3‐bisphophosphoglycerate (2,3‐BPG). Earlier studies demonstrated that erythrocyte 2,3‐BPG levels are elevated at a high altitude. However, what triggers its induction at high altitude is unknown. To address this question, we recruited 24 individuals and placed them at high altitude for different time points. Here we report that 1) Plasma adenosine and erythrocyte 2,3‐BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) their levels are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3‐DPG levels in normal individuals at high altitude. Next, we provide in vitro evidence that adenosine signaling via A2B receptor (ADORA2B) directly induced 2,3‐BPG production in a protein kinase A‐dependent manner in cultured human erythrocytes. Finally, we provide genetic and pharmacological evidence that that ADORA2B is essential for elevated adenosine induced 2,3‐BPG production and triggers O2 release in mice. Taken together, we reveal ADORA2B signaling cascade regulating erythrocyte 2,3‐BPG induction as a function of altitude and identify new targets to enhance O2 release under hypoxia conditions.Grant Funding Source: NIH

Evaluation of Oxidative Stress Status and Adenosine Deaminase Activity in Hyperthyroid and Hypothyroid Patients

A few prospective studies evaluated the oxidative stress variables and adenosine deaminase activity in patients with hyperthyroidism and hypothyroidism. Thus; we aimed to investigate oxidative stress variables and adenosine deaminase activity in patients with hyperthyroidism and hypothyroidism. Study populations consisted of 20 patients with hyperthyroidism, 20 patients with hypothyroidism and 20 age matched healthy volunteers as control groups. Plasma nitric oxide, malondialdehyde levels and glutathione, adenosine deaminase activities were measured in all subjects. plasma nitric oxide and adenosine deaminase levels were higher in patients with hyperthyroidism, but lower in patients with hypothyroidism compared to those of controls (P

The role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity.

Aim:We investigated the role of adenosine in citalopram-induced cardiotoxicity.Materials and Methods:Protocol 1: Rats were randomized into four groups. Sodium cromoglycate was administered to rats. Citalopram was infused after the 5% dextrose, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; A1 receptor antagonist), 8-(-3-chlorostyryl)-caffeine (CSC; A2a receptor antagonist), or dimethyl sulfoxide (DMSO) administrations. Protocol 2: First group received 5% dextrose intraperitoneally 1 hour prior to citalopram. Other rats were pretreated with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; inhibitor of adenosine deaminase) and S-(4-Nitrobenzyl)-6-thioinosine (NBTI; inhibitor of facilitated adenosine transport). After pretreatment, group 2 received 5% dextrose and group 3 received citalopram. Adenosine concentrations, mean arterial pressure (MAP), heart rate (HR), QRS duration and QT interval were evaluated.Results:In the dextrose group, citalopram infusion caused a significant decrease in MAP and HR and caused a significant prolongation in QRS and QT. DPCPX infusion significantly prevented the prolongation of the QT interval when compared to control. In the second protocol, citalopram infusion did not cause a significant change in plasma adenosine concentrations, but a significant increase observed in EHNA/NBTI groups. In EHNA/NBTI groups, citalopram-induced MAP and HR reductions, QRS and QT prolongations were more significant than the dextrose group.Conclusions:Citalopram may lead to QT prolongation by stimulating adenosine A1 receptors without affecting the release of adenosine.

Adenosine Is A Common Factor Regulating Erythrocyte 2,3-Bisphosphate Induction In Normal Individuals At High Altitude and In Patients With Sickle Cell Disease

To function effectively in O2 uptake, transport and delivery, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by endogenous allosteric modulators. One of the best know allosteric modulators is 2,3-bisphophosphoglycerate (2,3-BPG), a metabolic byproduct of glycolysis synthesized primarily in erythrocytes for the purpose of regulating Hb-O2 affinity. Earlier studies demonstrated that erythrocyte 2,3-BPG levels are elevated at a high altitude and in patients with sickle cell disease (SCD). We have recently shown that adenosine, a molecule well known to be induced under hypoxic conditions, is significantly elevated in SCD and contributes to increased 2,3-BPG induction in SCD erythrocytes. However, whether adenosine contributes to increased erythrocyte 2,3-BPG production at high altitude is unknown. To address this question we recruited 24 individuals who normally lived at sea level, and placed them at high altitude for different time points. Similarly, blood was collected from 45 SCD patients and 50 controls (at sea level). Here we report that 1) Plasma adenosine and erythrocyte 2,3-BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) The elevations of both molecules are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3-DPG levels in normal individuals at high altitude and in SCD patients. These results suggest that adenosine is a common factor responsible for elevated 2,3-BPG as a function in normal individuals and in SCD patients. To test this hypothesis we took pharmacologic and genetic approaches. We found that NECA, a stable adenosine analog, significantly increased 2,3-BPG production in cultured erythrocytes from normal and SCD mice and humans. Additional genetic and pharmacologic experiment showed that adenosine-induced 2,3-BPG production was mediated by ADORA2B receptor activation and downstream signaling by protein kinase A. To examine the in vivo physiological relevance of our findings we used adenosine deaminase (ADA)-deficient mice that accumulate high levels of circulating adenosine as a result of their enzyme deficiency. We found that elevated adenosine correlated to elevated erythrocyte 2,3-BPG and decreased Hb-O2 affinity. Experimental strategies to lower plasma adenosine or inhibit ADORA2B signaling in ADA-deficient mice resulted in reduced 2,3-BPG production and increased Hb-O2 affinity in erythrocytes. These studies provide in vivo evidence that elevated adenosine signaling via ADORA2B contributes to elevated of 2,3-BPG production and thereby triggers O2 release from erythrocytes. Taken together, our studies show that adenosine is a common factor regulating erythrocyte 2,3-BPG induction as a function of altitude and in patients with sickle cell disease. Our studies reveal numerous approaches to enhance oxygen release from oxyHb to physiologically or environmentally induced conditions of hypoxia. Disclosures:No relevant conflicts of interest to declare.

Plasma adenosine release is associated with bradycardia and transient loss of consciousness during experimental breath-hold diving

Plasma adenosine release is associated with bradycardia and transient loss of consciousness during experimental breath-hold diving Fabrice Joulia , Mathieu Coulange , Frederic Lemaitre , Guillaume Costalat , Frederic Franceschi , Vlad Gariboldi , Laetitia Nee , Julien Fromonot , Laurie Bruzzese , Gilles Gravier , Nathalie Kipson , Yves Jammes , Alain Boussuges , Michele Brignole , Jean Claude Deharo , Regis Guieu a,g,⁎

Abstract 341: Expression of Leukocyte CD39 in Acute Coronary Syndrome

Background CD39 (ENTPD-1) is a membrane-spanning ectonucleotidase that is responsible for the phosphohydrolysis of ATP to ADP and ADP to AMP. As increased myocardial infarct sizes have been reported in murine CD39 knockout models when compared with controls, we hypothesized that CD39 might be expressed as part of the myocardial injury/repair response. CD39 levels have not been heretofore described in humans with acute coronary syndrome (ACS). Objectives To measure CD39 mRNA and protein levels in human study subjects with ACS compared with healthy controls. Methods Healthy subjects (n=24), defined as having no history of coronary or peripheral arterial disease, and subjects with ACS (n=13) were enrolled in the study. Blood was collected via venupuncture for healthy controls or at the time of cardiac catheterization for ACS subjects. Plasma and leukocytes were isolated from blood, and soluble CD39 protein from leukocytes was measured using ELISA. mRNA was extracted from leukocytes, and qPCR was used to measure differences in leukocyte CD39 mRNA. Mass spec was used to assess plasma ATP, ADP, AMP, and adenosine levels. Findings Increased CD39 mRNA expression was found in subjects with ACS when compared with healthy controls (2.39 vs. 1.66, p=0.021). There was a trend towards increased leukocyte CD39 protein concentrations in ACS patients (n=5) compared to controls (n=9) (7.50 ng/ml vs. 2.64 ng/ml, p=0.057) when measured by ELISA. There was a trend toward increased levels of soluble plasma CD39 levels in ACS patients (n=12) when compared with controls (n=24) (2.39 ng/ml vs. 1.67 ng/ml, p=0.59) when measured by ELISA. ACS patients (n=11) were found to have higher levels of circulating ADP as assessed by Mass spec when compared with healthy controls (n=17), 2.02 vs. 0.67, p=0.03. Leukocyte CD39 levels positively correlated with ATP (r 2 =.231) and ADP (r 2 =.155) levels (n=10). Conclusions This is the first report of increased CD39 gene expression in patients with ACS, when compared with healthy controls without known coronary disease. A trend toward increased plasma and leukocyte CD39 protein expression was present in those with ACS when compared with controls. CD39 could serve as a modulator of vascular homeostasis in ACS.

Loss of equilibrative nucleoside transporter 1 in mice leads to progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis in humans

Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory spondyloarthropathy, characterized by ectopic calcification of spinal tissues. Symptoms include spine pain and stiffness, and in severe cases dysphagia and spinal cord compression. The etiology of DISH is unknown and there are no specific treatments. Recent studies have suggested a role for purine metabolism in the regulation of biomineralization. Equilibrative nucleoside transporter 1 (ENT1) transfers hydrophilic nucleosides, such as adenosine, across the plasma membrane. In mice lacking ENT1, we observed the development of calcified lesions resembling DISH. By 12 months of age, ENT1(-/-) mice exhibited signs of spine stiffness, hind limb dysfunction, and paralysis. Micro-computed tomography (µCT) revealed ectopic mineralization of paraspinal tissues in the cervical-thoracic region at 2 months of age, which extended to the lumbar and caudal regions with advancing age. Energy-dispersive X-ray microanalysis of lesions revealed a high content of calcium and phosphorus with a ratio similar to that of cortical bone. At 12 months of age, histological examination of ENT1(-/-) mice revealed large, irregular accumulations of eosinophilic material in paraspinal ligaments and entheses, intervertebral discs, and sternocostal articulations. There was no evidence of mineralization in appendicular joints or blood vessels, indicating specificity for the axial skeleton. Plasma adenosine levels were significantly greater in ENT1(-/-) mice than in wild-type, consistent with loss of ENT1--a primary adenosine uptake pathway. There was a significant reduction in the expression of Enpp1, Ank, and Alpl in intervertebral discs from ENT1(-/-) mice compared to wild-type mice. Elevated plasma levels of inorganic pyrophosphate in ENT1(-/-) mice indicated generalized disruption of pyrophosphate homeostasis. This is the first report of a role for ENT1 in regulating the calcification of soft tissues. Moreover, ENT1(-/-) mice may be a useful model for investigating pathogenesis and evaluating therapeutics for the prevention of mineralization in DISH and related disorders.

Search for adenosine A2A spare receptors on peripheral human lymphocytes

Some ligand–receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A2A receptors (A2AR), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A2AR remains to be established. We examined the situation using patients over-expressing lymphocyte A2AR and an agonist-like mAb to A2AR. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 μM, respectively). Lymphocyte A2AR expression was correlated to plasma adenosine level and A2AR occupation but not to A2AR response. These results are consistent with a lack of a reserve of functional A2AR on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A2AR determine the maximal level of the lymphocyte response to adenosine.

Elevated concentrations of plasma adenosine in obese children

There are no data regarding adenosine levels in obese children, even though is a ubiquitous molecule implicated in the regulation of lipid metabolism in humans. To determinate whether adenosine plasma levels are related with anthropometric and biochemical markers in children, we studied 51 students belong to Ramon Belmar School in Linares, Chile. Review of clinical data and frequent food questionnaire were taken in order to collect the information. Plasma adenosine levels were measured by high-performance liquid chromatography and biochemical parameters including insulin, glucose, total proteins, and lipid profile by using standard colorimetric assays. Children with detectable (above 0.1 μM) adenosine plasma levels (n = 30; BMI, 22.3 ± 0.7) had higher total cholesterol (P < 0.05); triglycerides (P < 0.01) and LDL-cholesterol (P < 0.05) concentrations than children with undetectable adenosine levels (n = 21; BMI, 23.9 ± 0.61). Among the analyzed variables, only BMI and BMI standard deviation score (BMI-SDS) were positively correlated with adenosine levels. Besides, obese children (n = 10) showed significantly high adenosine levels compared to controls (n = 11; 1.8 ± 0.2 vs. 1.2 ± 0.1 μM/mg protein, respectively. P < 0.05), but not compared to overweight children (n = 9). In conclusion, obesity in children is associated to high adenosine plasma levels. This study opens a new perspective to investigate the role of adenosine in the regulation of lipid metabolism in obese children.

Gestational Diabetes Reduces Adenosine Transport in Human Placental Microvascular Endothelium, an Effect Reversed by Insulin

Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44mapk) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44mapk/Akt ratios (‘metabolic phenotype’) were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44mapk/Akt ratios to normal pregnancies (‘mitogenic phenotype’). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM.