ATP Degradation Research Articles

Post mortem changes in M. iliotibialis lateralis muscle protein profile of emu (Dromaius novaehollandiae)

The available literature lacks information on the metabolic processes taking place in emu muscles after the cessation of circulation. Hence, this study was undertaken to examine the physicochemical characteristics (pH, drip loss, WHC, TBARS, L*, a*, b*) with concomitant changes in protein expression patterns (SDS-PAGE) of femoral muscle (M. iliotibialis lateralis) that occur post mortem and during the first days (0 h, 24 h, 48 h) of its maturation in 1- and 3-year-old emus. Our results indicated that the interaction between emus age and storage time had significant impact on meat pH and all color indicators. Furthermore, we detected 24 differentially expressed protein bands, representing 22 different gene products. ClueGO pathways analysis revealed that these proteins were mainly involved in glycolysis/gluconeogenesis pathway, pyruvate metabolism and pyrophosphate hydrolysis-driven proton transmembrane transporter activity. Based on the results obtained it can be assumed that early post-mortem metabolism of emu muscle is predominantly based on the glycolysis as reflected by the relative abundance alterations of the glycogenolytic and glycolytic enzymes. Moreover, the energy supplies provided by ATP and other high-energy substances degradation is higher in the group of older emus. Our findings also highlighted the complexity of the molecular mechanisms underlying the conversion of muscle to meat.

Pannexin 1 – a novel regulator of hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia by which pulmonary blood flow is redirected from poorly ventilated areas to better aerated lung regions. Sustained HPV plays a critical role in the development of pulmonary hypertension (PH) but the exact signaling pathways underlying HPV remain incompletely defined. Recently, ATP release via Pannexin 1 (Panx1) and subsequent purinergic signaling have been linked to transient receptor potential vanlloid 4 (TRPV4) channel mediated Ca2+ signaling in systemic vasoconstriction. Here, we probed for a similar role of Panx1 in HPV and the development of PH.To test the role of Panx1, we measured HPV as increase in pulmonary artery pressure (PAP) in response to hypoxia in lungs of endothelial cell (Cdh5-CreERT2/Panx1fl/fl) and smooth muscle cell specific (SMMHC-CreERT2/Panx1fl/fl) Panx1-KO mice. Isolated perfused lungs (IPL) of C57Bl6/J and Panx1-deficient mice were ventilated with normoxic (20% O2; 5% CO2, 75% N2) or hypoxic gas (1% O2; 5% CO2, 94% N2) and PAP was recorded via a pulmonary artery catheter. To elucidate the role of Panx1 in the development of PH, C57Bl6/J as well as Panx1-KO mice were kept under hypoxic conditions (10% O2) for 5 weeks followed by measurement of right ventricular systolic pressure, echocardiography and histology.Panx1 protein was expressed in human pulmonary artery endothelial cells as well as in human pulmonary artery smooth muscle cells (hPASMCs). Genetic deletion of Panx1 in smooth muscle cells, yet not in endothelial cells reduced the HPV response in isolated mouse lungs. In lungs of C57Bl6/J mice, Panx1-inhibition by the non-specific Panx1 inhibitors spironolactone or probenecid and the Panx1 specific inhibitory peptide (10Panx1) effectively attenuated HPV. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Interestingly, ATP release in response to hypoxia was not detectable in PASMC, and ATP degradation as well as inhibition of purinergic receptors failed to affect HPV. Rather, TRPV4 antagonism and Panx1 inhibition inhibited the hypoxia-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 signaling axis. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. Here we identify Panx1 as novel regulator of HPV. The role of Panx1 in HPV was, however, not attributable to ATP release and downstream purinergic receptor signaling or TRPV4 activation, but may involve a direct role of Panx1 as Ca2+ channel. In contrast to its role in HPV, Panx1 does not seem to contribute to chronic hypoxic PH.

Metabolic Abnormalities in Patients with Chronic Disorders of Consciousness.

The vegetative state (VS) and minimally conscious state (MCS) are two major types of chronic disorders of consciousness (DoC). The assessment of these two consciousness states generally relies on the Coma Recovery Scale-Revised (CRS-R) score, but a high misdiagnosis rate limits the generalized use of this score. To identify metabolites in human plasma that can accurately distinguish VS from MCS patients, comprehensive plasma metabolic profiles were obtained with targeted metabolomics analysis and untargeted and targeted lipidomics analysis. Univariate and multivariate analyses were used to assess the significance of differences. Compared with healthy controls (HCs), the DoC groups, Emerged from Minimally Conscious State (EMCS) group and Alzheimer’s disease (AD) group had significantly different metabolic profiles. Purine metabolism pathway differed the most between the DoC (MCS and VS) and HC groups. In this pathway, adenosine, ADP, and AMP, which are the derived products of ATP degradation, were decreased in the MCS and VS groups compared to healthy controls. More importantly, we identified certain lipids for which the levels were enriched in the VS or MCS groups. Specifically, phosphatidylcholine, (38:5)-H (PC(38:5)-H), and arachidonic acid (AA) differed substantially between the VS and MCS groups and may be used to distinguish these two groups of patients. Together, our findings suggest that metabolic profiling is significantly altered in patients with chronic DoC.

The Protective Role of the Carbohydrate Response Element Binding Protein in the Liver: The Metabolite Perspective

The Carbohydrate response element binding protein, ChREBP encoded by the MLXIPL gene, is a transcription factor that is expressed at high levels in the liver and has a prominent function during consumption of high-carbohydrate diets. ChREBP is activated by raised cellular levels of phosphate ester intermediates of glycolysis, gluconeogenesis and the pentose phosphate pathway. Its target genes include a wide range of enzymes and regulatory proteins, including G6pc, Gckr, Pklr, Prkaa1,2, and enzymes of lipogenesis. ChREBP activation cumulatively promotes increased disposal of phosphate ester intermediates to glucose, via glucose 6-phosphatase or to pyruvate via glycolysis with further metabolism by lipogenesis. Dietary fructose is metabolized in both the intestine and the liver and is more lipogenic than glucose. It also induces greater elevation in phosphate ester intermediates than glucose, and at high concentrations causes transient depletion of inorganic phosphate, compromised ATP homeostasis and degradation of adenine nucleotides to uric acid. ChREBP deficiency predisposes to fructose intolerance and compromised cellular phosphate ester and ATP homeostasis and thereby markedly aggravates the changes in metabolite levels caused by dietary fructose. The recent evidence that high fructose intake causes more severe hepatocyte damage in ChREBP-deficient models confirms the crucial protective role for ChREBP in maintaining intracellular phosphate homeostasis. The improved ATP homeostasis in hepatocytes isolated from mice after chronic activation of ChREBP with a glucokinase activator supports the role of ChREBP in the control of intracellular homeostasis. It is hypothesized that drugs that activate ChREBP confer a protective role in the liver particularly in compromised metabolic states.

New contributions to the relationship between sequential changes of ATP-related metabolites and post-mortem interval in rats.

In recent years, it has become a new research direction to post-mortem interval (PMI) estimation from the changes in metabolite content generated during the process of cadaver corruption, and lead to more and more attention has been paid to the biochemical changes of muscles after death. The ATP degradation process has long been accepted as a precise manner to estimate freshness of meat products in food science. ATP-related metabolites may also serve as an indicator for PMI estimation of corpses. The purpose of this study was to test this possibility. Firstly, this study optimized the detection method of ATP-related metabolites in skeletal muscles of rat. Moreover, in animal experiment, ATP-related metabolites (ATP, ADP, AMP, IMP, Ino, Hx) in lower hind limb skeletal muscles of sixty rats were measured by RP-HPLC at different PMI for 168h at 20°C±1°C, the change of freshness indicators (K value, K1 value, K0 value, H value, P value, G value, IMP ratio) were carefully analyzed, and investigate the relationship between them and PMI. The overall results showed that IMP, AMP, Hx, Total metabolites, K value, K1 value, K0 value, H value, P value, G value, and IMP ratio in skeletal muscles changed significantly with PMI (R2=0.864-0.971, all P<0.01) while ATP, ADP, Ino, IMP precursors and IMP degradants had minor changes during the same period (R2=0.171-0.706). Specifically, significant linear positive correlations between H value of skeletal muscles and PMI were found, and the coefficients of their regression functions were R2=0.971. The correlation between AMP and PMI was slightly lower (R2=0.864), but the scope of PMI estimation was the widest (168h). It can be concluded that the determination of ATP-related metabolites in skeletal muscle may be a potential tool in the PMI estimation. However, more researches on its influencing factors are needed to facilitate its final use in practice.

Xanthine oxidase inhibition attenuates doxorubicin-induced cardiotoxicity in mice

Abstract Background Accumulating evidence suggests that high serum uric acid (UA) is associated with left ventricular (LV) dysfunction. Although xanthine oxidase (XO) activation is a critical regulatory mechanism of the terminal step in ATP and purine degradation, the pathophysiological role of cardiac tissue XO in LV dysfunction remains unclear. Objectives We hypothesized that cardiac XO is activated in doxorubicin-induced LV dysfunction, and XO inhibitors ameliorate LV function by inhibiting cell death signals as well as by modifying cardiac purine metabolism. Methods Either doxorubicin (10 mg/kg) or vehicle was intraperitonially administered in a single injection to ICR mice. Mice were treated with or without oral XO inhibitors (febuxostat 3 mg/kg/day or topiroxostat 5 mg/kg/day) for 8 days starting 24 hours before doxorubicin-injection. The LV function was assessed by echocardiography at day 6 and by ex vivo heart perfusion at day 7. Results Cardiac tissue XO activity measured by a highly sensitive assay with liquid chromatography/mass spectrometry (n=8 each) and cardiac UA content (n=3–6) were significantly increased in doxorubicin-treated mice at day 7 and dramatically reduced by XO inhibitors. Accordingly, XO inhibitors substantially improved LV ejection fraction (n=8 each) and LV developed pressure (n=9 each) that had been impaired by doxorubicin administration. Intriguingly, the expression of GPX4, a negative regulator of ferroptosis, was decreased in doxorubicin-treated hearts but improved by XO inhibitors (n=6 each). Furthermore, metabolome analyses revealed an enhanced purine metabolism in doxorubicin-treated hearts, and XO inhibitors suppressed the serial metabolic reaction of hypoxanthine–xanthine–UA. Conclusions Doxorubicin administration induces cardiac tissue XO activation associated with an impaired LV function. XO inhibition attenuates the doxorubicin-induced cardiotoxicity partly through an anti-ferroptotic effect and the conservation of tissue ATP levels by modulating purine metabolism. The present study suggests that pharmacological XO inhibition represents a potential therapeutic strategy for the treatment of doxorubicin-induced cardiotoxicity. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This study was supported in part by grants-in-aid for Ministry of Education Culture, Sports, Science and Technology.

Xanthine oxidase inhibition attenuates doxorubicin-induced cardiotoxicity in mice

Accumulating evidence suggests that high serum uric acid (UA) is associated with left ventricular (LV) dysfunction. Although xanthine oxidase (XO) activation is a critical regulatory mechanism of the terminal step in ATP and purine degradation, the pathophysiological role of cardiac tissue XO in LV dysfunction remains unclear. We herein investigated the role and functional significance of tissue XO activity in doxorubicin-induced cardiotoxicity. Either doxorubicin (10 mg/kg) or vehicle was intraperitonially administered in a single injection to mice. Mice were treated with or without oral XO-inhibitors (febuxostat 3 mg/kg/day or topiroxostat 5 mg/kg/day) for 8 days starting 24 h before doxorubicin injection. Cardiac tissue XO activity measured by a highly sensitive assay with liquid chromatography/mass spectrometry and cardiac UA content were significantly increased in doxorubicin-treated mice at day 7 and dramatically reduced by XO-inhibitors. Accordingly, XO-inhibitors substantially improved LV ejection fraction (assessed by echocardiography) and LV developed pressure (assessed by ex vivo Langendorff heart perfusion) impaired by doxorubicin administration. This was associated with an increase in XO-derived hydrogen peroxide production with concomitant upregulation of apoptotic and ferroptotic pathways, all of which were reduced by XO-inhibitors. Furthermore, metabolome analyses revealed enhanced purine metabolism in doxorubicin-treated hearts, and XO-inhibitors suppressed the serial metabolic reaction of hypoxanthine–xanthine–UA, the paths of ATP and purine degradation. In summary, doxorubicin administration induces cardiac tissue XO activation associated with impaired LV function. XO-inhibitors attenuate doxorubicin-induced cardiotoxicity through inhibition of XO-derived oxidative stress and cell death signals as well as the maintenance of cardiac energy metabolism associated with modulation of the purine metabolic pathway.

Extracellular ATP Increases Glucose Metabolism in Skeletal Muscle Cells in a P2 Receptor Dependent Manner but Does Not Contribute to Palmitate-Induced Insulin Resistance.

Saturated fatty acids such as palmitate contribute to the development of Type 2 Diabetes by reducing insulin sensitivity, increasing inflammation and potentially contributing to anabolic resistance. We hypothesized that palmitate-induced ATP release from skeletal muscle cells may increase inflammatory cytokine production and contribute to insulin/anabolic resistance in an autocrine/paracrine manner. In C2C12 myotubes differentiated at physiological glucose concentrations (5.5 mM), palmitate treatment (16 h) at concentrations greater than 250 μM increased release of ATP and inflammatory cytokines IL-6 and MIF, significantly blunted insulin and amino acid-induced signaling and reduced mitochondrial function. In contrast to our hypothesis, degradation of extracellular ATP using apyrase, did not alter palmitate-induced insulin resistance nor alter release of cytokines. Moreover, treatment with ATPγS (16 h), a non-hydrolysable ATP analog, in the absence of palmitate, did not diminish insulin sensitivity. Acute treatment with ATPγS produced insulin mimetic roles; increased phosphorylation of PKB (aka AKT), S6K1 and ERK and enhanced GLUT4-mediated glucose uptake in the absence of exogenous insulin. The increases in PKB and S6K1 phosphorylation were completely prevented by pre-incubation with broad spectrum purinergic receptor (P2R) blockers PPADs and suramin but not by P2 × 4 or P2 × 7 blockers 5-BDBD or A-438079, respectively. Moreover, ATPγS increased IL-6 yet decreased MIF release, similar to the cytokine profile produced by exercise. Acute and chronic treatment with ATPγS increased glycolytic rate in a manner that was differentially inhibited by PPADs and suramin, suggesting heterogeneous P2R activation in the control of cellular metabolism. In summary, our data suggest that the palmitate-induced increase in ATP does not contribute to insulin/anabolic resistance in a cell autonomous manner.

Deletion of TRPV4 enhances in vitro wound healing of murine esophageal keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that is widely expressed in different body tissues and plays several physiological roles. This channel is highly expressed in esophageal keratinocytes where its activation mediates ATP release. However, whether TRPV4 has a role in wound healing of esophageal keratinocytes is unclear. In this study, we demonstrated that both cell migration and proliferation were slower in wild-type esophageal keratinocytes compared to cells having TRPV4 knockout. Our results suggest that TRPV4-mediated release of ATP from esophageal keratinocytes contributes to a decrease in the rate of in vitro wound healing via the ATP degradation product adenosine, which acts on A2B adenosine receptors.

Comparison of Detection Limits for Allergenic Foods between Total Adenylate (ATP+ADP+AMP) Hygiene Monitoring Test and Several Hygiene Monitoring Approaches

Validation and verification of cleaning and inspection methods are essential to prevent the spread of allergens via cross-contact. Among the hygiene monitoring tests used on-site, the ATP test is rapid and provides quantifiable results. Nevertheless, because a wide variety of foods contain significant amount of ADP and/or AMP due to the degradation of ATP, the ATP+ADP+AMP (A3) test is preferred for detecting food debris. Hence, the A3 test may be valuable in screening food debris that may contain residual allergens. In this study, the detection limits of the A3 test for 40 foods that are regulated in several countries as allergenic were compared with those of the other hygiene monitoring tests used on-site: the conventional ATP test with similar sensitivity for ATP, the protein swab test that detects as little as 50 μg of protein, and the lateral flow immunoassay (LFI). The A3 test demonstrated lower detection limits than did the ATP test. The detection sensitivity of the A3 test was greater than that of the protein swab test except for its use on gelatin (extracted protein). The cleaning validation performance using a stainless steel model in fish and meat revealed that the A3 test is efficient in verifying the levels of remaining food debris. Although LFI displayed the best sensitivities for 10 of 14 foods, it is not commercially available for some specific allergens; however, the A3 test can detect such food debris. Moreover, the detection limits of the A3 test were preferable or comparable to those of LFI for crustacean shellfish and for processed grains, with the exception of wheat flour and buckwheat. A field study in a food processing plant demonstrated that the amount of both A3 and milk protein (enzyme-linked immunosorbent assay) considerably decreased as the cleaning steps progressed. Therefore, the A3 test is effective in detecting the risk for allergen cross-contact after inadequate cleaning.

Asthmatic airway inflammation was alleviated by limiting the level of lung extracellular ATP

Abstract Asthma is a chronic respiratory disease characterized by recurrently attacks of breathlessness and wheezing. ATP accumulation in bronchoalveolar lavage fluid (BALF) of asthmatic subjects indicates that extracellular ATP (eATP) and downstream responses are involved. The level of eATP is tightly controlled by its release and degradation in healthy tissues. Pannexin-1 is a channel contributing to ATP release, and CD39 is membrane bound apyrase contributing to ATP degradation. Cellular stress or inflammatory stimulation leads increased release of ATP and accompanied with the reduction of CD39. The recombinant adeno-associated virus (rAAV) vectors have been developed for efficient gene transfer with low toxic and difference tissue tropisms. Thus, we generated rAAV-CD39 and rAAV-s10Panx1 and examined their efficacy in OVA-mediated asthmatic mice model. The mice were sensitized intraperitoneally and challenged intratracheally with OVA and treated with the vectors three days before OVA challenge. At the end of procedure, some asthmatic and inflammatory cardinal features were examined. Elevated ATP level either in stimulated-culture supernatant and BALF of OVA-sensitized mice was determined. These two treatments downregulated the levels of ATP in BALF. Several asthmatic features, such as eosinophilia, Th2 cytokine production and AHR were decreased in the treatment groups. These treatments altered the T cell response by targeting the danger signal eATP and its metabolites. Taken together, these approaches are able to downregulate the asthma symptoms with the reduction of inflammatory responses in the lungs. Based on these results, eATP can be considered as a potential gene therapy target for asthma.

Targetting E.coli growth in vivo in intervention against urinary tract infections

UTI is an everyday encounter in the primary health sector, and in the US, UTI accounts for 8.1% of all hospital admissions 1. In Denmark, with 5.6 mill citizens, over 500,000 prescriptions of antibiotics for UTIs are issued per year 2and even in the primary sector, one finds a high incidence of antibiotic resistance in bacteria that cause UTI 3. This stresses the need for new approaches to supplement standard antibiotic treatment to control UTIs. UTIs are most frequently caused by E. coli that produce a variety of virulence factors 4– 8, of which α‐haemolysin (HlyA) is the most consistent. Our previous in vitro studies demonstrate that the biological effects of HlyA are to a large extent are secondary to extracellular ATP signalling. Interestingly, insertion of HlyA into a cells membrane inflicts non‐lytic ATP release from cells directly through the HlyA‐pore. In our murine model of ascending UTI, it is easily confirmed that HlyA is a virulence factor that promotes development of pyelonephritis and we have shown that HlyA is responsible for the majority of the septic symptoms in a murine model of urosepsis. Therefore, we hypothesise that release of host ATP may be essential for the virulence promoted by HlyA.We test the hypothesis by a combination of bacterial growth studies (flow cytometry and OD660) and in vivo murine model of pyelonephritis. Our data show that E.coli grow exceedingly well in urine from healthy volunteers, despite its low iron content17. Within the first couple of hours, the growth rate of E.coli is significantly higher in urine than in Lysogeny Broth (LB). In contrast, the E.coli practically does not grow in physiological saline (HEPES buffered salt solution, HBS) containing 5.6 mM glucose. Moreover, we found that E.coli grows faster in hyperosmotic compared to dilute urine and addition of as little as 25μl human urine per ml HBS was sufficient to stimulate E.coli growth maximally. This supports that the initial accelerated growth in urine compared to LB medium must be a substance found explicitly in the urine. By screening, we exclude urea, iron, creatinine, proteins/polypeptides and taurin to have any effect on bacterial growth. We did, however, find that NH4+ was necessary as a nitrogen donor. Moreover, it was observed that ATP degradation products ADP and AMP, which are present in the urine in micromolar concentrations, markedly supported and accelerated E. coli growth in HBS. Our in vitro data signifies that dilution of the urine potentially could protect against ascending UTI and our initial in vivo studies of ascending UTI in mice support that the urine has to be diluted to values below 300 mosmol/l to be able to detect this effect. Therefore, simple dilution would not provide sufficient protection against ascending UTIs. Consequently, we are currently specifically targeting the ADP/AMP and NH4+ pathways in our strive to develop new treatment strategies for prevention of UTI.Support or Funding InformationThis project was funden byThe Independent Research fund ‐DenmarkThe Augustinus Foundation

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists of treatment with imatinib mesylate, which selectively inhibits the BCR-ABL protein by competing for its ATP-binding site. Adenine nucleotide signaling is modulated by the ectonucleotidases and this pathway is related to tumorigenic processes. Considering the relationship between ATP and cancer, we aimed to evaluate the influence of imatinib mesylate on the expressions and functions of the NTPDase and ecto-5'-nucleotidase (CD73) enzymes in imatinib-sensitive and -resistant K-562 cell lines. mRNA analysis showed that K-562 cells express all ENTPDs and NT5E. However, when treated with imatinib mesylate for 24h, the expression of ENTPD1, -2, -3 and -5 increased, leading to a higher nucleotides hydrolysis rate. HPLC analysis identified increased ATP degradation in cells after 24h of treatment, with consequent ADP and AMP formation, corroborating the increase in gene and protein expression of ectonucleotidases as observed in previous results. On the other hand, we observed that imatinib-resistant K-562 cells presented a decrease in nucleotide hydrolysis and expressions of ENTPD1 and -5. These results suggest an involvement of imatinib in modulating ectonucleotidases in CML that will need further investigation. Since these ectonucleotidases have important catalytic activities in the tumor microenvironment, their modulation in CML cells may represent an important therapeutic approach to regulate levels of extracellular adenine nucleotides.

Increased host ATP efflux and its conversion to extracellular adenosine is crucial for establishing Leishmania infection.

Intracellular survival of Leishmania donovani demands rapid production of host ATP for its sustenance. However, a gradual decrease in intracellular ATP in spite of increased glycolysis suggests ATP efflux during infection. Accordingly, upon infection, we show here that ATP is exported and the major exporter was pannexin-1, leading to raised extracellular ATP levels. Extracellular ATP shows a gradual decrease after the initial increase, and analysis of cell surface ATP-degrading enzymes revealed induction of the ectonucleotidases CD39 and CD73. Ectonucleotidase-mediated ATP degradation leads to increased extracellular adenosine (eADO), and inhibition of CD39 and CD73 in infected cells decreased adenosine concentration and parasite survival, documenting the importance of adenosine in infection. Inhibiting adenosine uptake by cells did not affect parasite survival, suggesting that eADO exerts its effect through receptor-mediated signalling. We also show that Leishmania induces the expression of adenosine receptors A2AR and A2BR, both of which are important for anti-inflammatory responses. Treating infected BALB/c mice with CD39 and CD73 inhibitors resulted in decreased parasite burden and increased host-favourable cytokine production. Collectively, these observations indicate that infection-induced ATP is exported, and after conversion into adenosine, propagates infection via receptor-mediated signalling.

In Memoriam Geoffrey Burnstock: Creator of Purinergic Signaling

Geoff Burnstock (1929–2020) discovered purinergic signaling in a fastidious research that started in early 1960 and culminated in a concept of purinergic nerves in 1972. Subsequently, Geoff developed the concept of purinergic transmission and demonstrated ATP storage, release, and degradation in the context of cotransmission, which was another fundamental concept developed by him. Purinergic transmission contributes to the most fundamental physiological functions such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, as well as to various pathophysiological conditions, including inflammation, cancer, neuropathic pain, diabetes, and kidney failure.

Adenosine Generated by Regulatory T Cells Induces CD8+ T Cell Exhaustion in Gastric Cancer through A2aR Pathway.

Background Adenosine, derived from the degradation of ATP via ectonucleotidases CD39 and CD73, is a critical immunosuppressive metabolite in the hypoxic microenvironment of tumor tissue. Adenosine signaling via A2aR can inhibit the antitumor immune response of CD8+ T cells. CD39 and CD73 high-expressing Tregs play a critical role in tumor immune evasion of gastric cancer (GC). The present study investigated the underlying mechanism by which Tregs suppress antitumor immune responses in GC. Materials and Methods Fifty-two GC samples were collected, and the frequency of FoxP3+ Tregs and CD8+ T cells and density ratios of A2aR+/CD8+ T cells, CD39+/FoxP3+ Tregs, and CD73+/FoxP3+ Tregs in GC were assessed with multiplex immunofluorescence. The expression of FoxP3 and A2aR in GC tissues was also detected by the immunoblotting assay. We next investigated the relationship between density of FoxP3+ Tregs, ratio of A2aR+/CD8+ T cells, and clinicopathological parameters. At the same time, Tregs and CD8+ T cells were isolated from peripheral blood of five GC patients, and the antagonists of CD39 and CD73 were used to assess the ability of Tregs to decompose ATP into adenosine. In addition, we cocultured CD8+ T cells and Tregs with antagonists of A2aR and A2bR in order to examine the alterations in immune function of CD8+ T cells. Results The density of both FoxP3+ Tregs and A2aR+/CD8+ T cells was higher in GC tissue compared to peritumoral normal tissue and significantly correlated with the TNM stage, lymph node metastasis, and distant metastasis of GC. The process of Treg hydrolysis of ATP into adenosine was blocked by the antagonists of CD39 and CD73. In addition, Tregs could induce apoptosis and inhibit proliferation of CD8+ T cells, while this effect could be obviously reduced by applying the antagonist of A2aR or A2aR+A2bR. Moreover, IFN-γ, TNF-α, and perforin generated by CD8+ T cells could also be inhibited through the adenosine A2aR pathway. Conclusions The FoxP3+ Tregs and A2aR+/CD8+ T cells were excessively infiltrated in GC tissue. Tregs from GC can decompose ATP to adenosine and in turn induce apoptosis and inhibit the proliferation of CD8+ T cells through the A2aR pathway, further leading to immune escape of GC.

The fate of acetamiprid and its degradation during long-term storage of honey

ABSTRACTAcetamiprid (ATP) is a neonicotinoid widely used in agriculture, which is less toxic to honeybees and is transported into the hives via the contaminated nectar and pollen as well as physical contact. Although ATP degrades in animal systems mainly through N-desmethylacetamiprid (IM-2-1), it is degraded in honey of different origins mainly through (E)-N2-carbamoyl-)-N1-[6-chloro-3-pyridyl)methyl])-N1-methylacetamidine (IM-1-2). The decomposition of ATP in honey has been followed for storage times up to 180 days at 20°C, 30°C and 40°C to represent geographical regions with different temperatures. Subsequent degradation of IM-1-2 occurred and IM-1-4 was detected as a degradation product of IM-1-2. The apparent decomposition rate constants and half-lives of ATP in honey were determined. ATP degradation to IM-1-2 was faster in honey than the rates observed in aqueous solutions of pH 3.0–5.0. In order to follow actual ATP contamination in honey, IM-1-2 analysis is proposed to be carried out instead of the analyses of ATP and IM-2-1 for safety determination to ensure application of Good Agricultural Practice and to maintain a healthy environment.

Sensory and ATP derivative-based indicators for assessing the freshness of Atlantic salmon (Salmo salar) and cod (Gadus morhua)

Abstract To estimate the shelf life of fresh fish, the processor must know the period of time between catch/harvest and arrival at the processing plant. This information is not always available, necessitating the provision of methods to estimate the time since catch or harvest. The objectives of this study were therefore to develop and/or validate sensory and ATP derivative-based methods for rapidly assessing the freshness of fish. A quality index method (QIM; raw fish) and a quantitative descriptive analysis (QDA; cooked fish) were developed and validated (against bacterial count [total viable count (TVC)] and time) for salmon (Salmo salar) and cod (Gadus morhua). The production of inosine monophosphate (IMP), inosine and hypoxanthine (Hx) and associated ratios (IMP/Hx, K1-value or H-value) were also investigated for use as freshness markers. There was a linear relationship between QIM and TVC (R2 = 0.93 for salmon and R2 = 0.89 for cod), QIM and time (R2 = 0.96 for salmon and R2 = 0.98 for cod), QDA and TVC (R2 = 0.93 for salmon and R2 = 0.77 for cod) and QDA and time (R2 = 0.94 for salmon and R2 = 0.87 for cod), suggesting that the QIM and QDA schemes developed could be used to monitor/assess freshness. The H-value also increased linearly with TVC (R2 = 0.88 for salmon and R2 = 0.89 for cod) and time (R2 = 0.93 for salmon and R2 = 0.84 for cod). It was therefore concluded that both the QIM/QDA approach and monitoring ATP degradation, specifically expressed as the H-value, could be used as rapid methods to assess the freshness of salmon and cod arriving at the processing plant.

Genome-wide identification, characterization and expression pattern analysis of APYRASE family members in response to abiotic and biotic stresses in wheat.

APYRASEs, which directly regulate intra- and extra-cellular ATP homeostasis, play a pivotal role in the regulation of various stress adaptations in mammals, bacteria and plants. In the present study, we identified and characterized wheat APYRASE family members at the genomic level in wheat. The results identified a total of nine APY homologs with conserved ACR domains. The sequence alignments, phylogenetic relations and conserved motifs of wheat APYs were bioinformatically analyzed. Although they share highly conserved secondary and tertiary structures, the wheat APYs could be mainly categorized into three groups, according to phylogenetic and structural analysis. Additionally, these APYs exhibited similar expression patterns in the root and shoot, among which TaAPY3-1, TaAPY3-3 and TaAPY3-4 had the highest expression levels. The time-course expression patterns of the eight APYs in response to biotic and abiotic stress in the wheat seedlings were also investigated. TaAPY3-2, TaAPY3-3, TaAPY3-4 and TaAPY6 exhibited strong sensitivity to all kinds of stresses in the leaves. Some APYs showed specific expression responses, such as TaAPY6 to heavy metal stress, and TaAPY7 to heat and salt stress. These results suggest that the stress-inducible APYs could have potential roles in the regulation of environmental stress adaptations. Moreover, the catalytic activity of TaAPY3-1 was further analyzed in the in vitro system. The results showed that TaAPY3-1 protein exhibited high catalytic activity in the degradation of ATP and ADP, but with low activity in degradation of TTP and GTP. It also has an extensive range of temperature adaptability, but preferred relatively acidic pH conditions. In this study, the genome-wide identification and characterization of APYs in wheat were suggested to be useful for further genetic modifications in the generation of high-stress-tolerant wheat cultivars.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.