The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation.
Ontology highlight
ABSTRACT:
SUBMITTER: Newby AC
PROVIDER: S-EPMC1149266 | biostudies-other | 1988 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.
| S-EPMC1148762 | biostudies-other
Project description:Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.
| S-EPMC1138859 | biostudies-other
Project description:A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.
| S-EPMC1131399 | biostudies-other
Project description:BackgroundAdenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5'-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5'-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73-/- and wild type (WT) mice.Methods and results3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.ConclusionThe infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5'-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine's well known cardioprotective effect in early phase ischemic preconditioning.
| S-EPMC4532361 | biostudies-literature
Project description:Heart rates during ischaemia and reperfusion are possible determinants of reperfusion arrhythmias. We used ivabradine, a selective If current inhibitor, to assess the effects of heart rate reduction (HRR) during ischaemia-reperfusion on reperfusion ventricular arrhythmias and assessed potential anti-arrhythmic mechanisms by optical mapping. Five groups of rat hearts were subjected to regional ischaemia by left anterior descending artery occlusion for 8min followed by 10min of reperfusion: (1) Control n=10; (2) 1?M of ivabradine perfusion n=10; (3) 1?M of ivabradine+5Hz atrial pacing throughout ischaemia-reperfusion n=5; (4) 1?M of ivabradine+5Hz pacing only at reperfusion; (5) 100?M of ivabradine was used as a 1ml bolus upon reperfusion. For optical mapping, 10 hearts (ivabradine n=5; 5Hz pacing n=5) were subjected to global ischaemia whilst transmembrane voltage transients were recorded. Epicardial activation was mapped, and the rate of development of ischaemia-induced electrophysiological changes was assessed. HRR observed in the ivabradine group during both ischaemia (195±11bpm vs. control 272±14bpm, p<0.05) and at reperfusion (168±13bpm vs. 276±14bpm, p<0.05) was associated with reduced reperfusion ventricular fibrillation (VF) incidence (20% vs. 90%, p<0.05). Pacing throughout ischaemia-reperfusion abolished the protective effects of ivabradine (100% VF), whereas pacing at reperfusion only partially attenuated this effect (40% VF). Ivabradine, given as a bolus at reperfusion, did not significantly affect VF incidence (80% VF). Optical mapping experiments showed a delay to ischaemia-induced conduction slowing (time to 50% conduction slowing: 10.2±1.3min vs. 5.1±0.7min, p<0.05) and to loss of electrical excitability in ivabradine-perfused hearts (27.7±4.3min vs. 14.5±0.6min, p<0.05). Ivabradine administered throughout ischaemia and reperfusion reduced reperfusion VF incidence through HRR. Heart rate during ischaemia is a major determinant of reperfusion arrhythmias. Heart rate at reperfusion alone was not a determinant of reperfusion VF, as neither a bolus of ivabradine nor pacing immediately prior to reperfusion significantly altered reperfusion VF incidence. This anti-arrhythmic effect of heart rate reduction during ischaemia may reflect slower development of ischaemia-induced electrophysiological changes.
| S-EPMC3654199 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Inactivation of the gene for adenosine A2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell-specific pathogenic effects of A2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A2A receptors after thromboembolic stroke improves post-stroke outcomes via down-regulation of inflammation. EXPERIMENTAL APPROACH:Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post-stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood-brain barrier (BBB) leakage, and oedema assessment. Anti-inflammatory effects of silencing the gene for A2A receptors or pharmacological antagonism of these receptors were assessed in vitro. KEY RESULTS:Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a-/- ) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2a?VEC ) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down-regulating cleaved caspase 1 and IL-1? expression. CONCLUSIONS AND IMPLICATIONS:Specific inactivation of endothelial A2A receptors mitigated ischaemic brain injury and improved post-stroke outcomes, at least partly, through anti-inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke.
| S-EPMC6555863 | biostudies-literature
Project description:To investigate the broad transcriptional consequences of Nt5e knock-out on mice vascular smooth cells in culture at baseline.
2022-11-01 | GSE216727 | GEO
Project description:Background and purposeCytochrome c when released from mitochondria into cytosol triggers assembly of the apoptosome resulting in caspase activation. Recent evidence suggests that reduced cytochrome c is unable to activate the caspase cascade. In this study, we investigated whether a chemical reductant of cytochrome c, N,N,N',N'-tetramethylphenylene-1,4-diamine (TMPD), which we have previously shown to block cytochrome c-induced caspase activation, could prevent ischaemia-induced apoptosis in the rat perfused heart.Experimental approachThe Langendorff-perfused rat hearts were pretreated with TMPD and subjected to stop-flow ischaemia or ischaemia/reperfusion. The activation of caspases (measured as DEVD-p-nitroanilide-cleaving activity), nuclear apoptosis of cardiomyocytes (measured by dUTP nick end labelling assay), mitochondrial and cytosolic levels of cytochrome c (measured spectrophotometrically and by elisa), and reperfusion-induced necrosis (measured as the activity of creatine kinase released into perfusate) were assessed.Key resultsWe found that perfusion of the hearts with TMPD strongly inhibited ischaemia- or ischaemia/reperfusion-induced activation of caspases and partially prevented nuclear apoptosis in cardiomyocytes. TMPD did not prevent ischaemia- or ischaemia/reperfusion-induced release of cytochrome c from mitochondria into cytosol. TMPD also inhibited ischaemia/reperfusion-induced necrosis.Conclusions and implicationsThese results suggest that TMPD or related molecules might be used to protect the heart against damage induced by ischaemia/reperfusion. The mechanism of this protective effect of TMPD probably involves electron reduction of cytochrome c (without decreasing its release) which then inhibits the activation of caspases.
| S-EPMC3051385 | biostudies-literature
Project description:Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5'-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado).
| S-EPMC4570785 | biostudies-literature
Project description:Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood.We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression.Using high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5'-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II-infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II-induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-?-dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-? is an important factor responsible for angiotensin II-induced CD73 and ADORA2B expression at the transcriptional level.Overall, our studies reveal that angiotensin II-induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling-mediated endothelin-1 induction in a hypoxia-inducible factor-?-dependent manner. The inhibition of excess adenosine-mediated ADORA2B signaling represents a novel therapeutic target for the disease.
| S-EPMC3886128 | biostudies-literature