scholarly journals Extracellular Degradation into Adenosine and the Activities of Adenosine Kinase and AMPK Mediate Extracellular NAD+-produced increases in the Adenylate Pool of BV2 Microglia under Basal Conditions

2018 ◽  
Author(s):  
Jie Zhang ◽  
Caixia Wang ◽  
Danhong Wu ◽  
Weihai Ying
Keyword(s):  
Adenosine Kinase ◽  
Protective Effects ◽  
Physiological Conditions ◽  
Equilibrative Nucleoside Transporters ◽  
Bv2 Microglia ◽  
Adenylate Pool ◽  
Aging Models ◽  
Nanomolar Range ◽  
Fof1 Atp Synthase ◽  
Key Questions

AbstractCumulating evidence has indicated NAD+ deficiency as a common central pathological factor of multiple diseases and aging. NAD+ supplement is highly protective in various disease and aging models, while two key questions remain unanswered: 1) Does extracellular NAD+ also produce its effects through its degradation product adenosine? 2) Does extracellular NAD+ produce the protective effects by affecting cells under pathological insults only, or by affecting both normal cell and cells under pathological insults? Since extracellular NAD+ can be degraded into adenosine, and endogenous adenosine levels are in the nanomolar range under physiological conditions, extracellular NAD+ may produce its effects through its degradation into adenosine. In this study we used BV2 microglia as a cellular model to test our hypothesis that NAD+ treatment can increase the intracellular adenylate pool under basal conditions through its extracellular degradation into adenosine. Our study has shown that extracellular NAD+ increases the adenylate pool of BV2 microglia under basal conditions through its degradation into adenosine that enters the cells through equilibrative nucleoside transporters. The intracellular adenosine is converted to AMP by adenosine kinase, which increases intracellular ATP by both activating AMPK and increasing ADP that drives mitochondrial FoF1-ATP synthase. Collectively, our study has suggested that extracellular NAD+ can enhance defensive capacity of normal cells through a novel pathway, which includes extracellular NAD+ degradation into adenosine and the activities of adenosine kinase and AMPK. Our findings have also suggested that NAD+ administration in various disease and aging models may significantly affect the microglia under basal conditions.

scholarly journals In Vivo Enzymes Activities of Some Ru(II) Compounds with N-Alkylphenothiazines

2016 ◽  
Vol 66 (4) ◽  
pp. 497-508
Author(s):  
P. Milena Krstić ◽  
Z. Sunčica Borozan ◽  
P. Sofija Sovilj ◽  
R. Sanja Grgurić-Šipka ◽  
M. Jelena Oljarević
Keyword(s):  
Antioxidant Activity ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Normal Activity ◽  
Protective Effects ◽  
Toxic Compounds ◽  
Physiological Conditions ◽  
Positive Effects ◽  
Ldh Activity

Abstract The purpose of the present study was to investigate and compare the effects of two ruthenium complexes with trifluoperazine on acethylcholinesterase enzyme activity and lactate dehydrogenase levels in vivo under physiological conditions in rats blood. Complexes 1 and 2 showed positive effects on acethylcholinesterase at all doses and did not disturb its normal activity. Total LDH activity was inhibited in the presence of both complexes, but Ru(II) complexes showed different effects on the activity of LDH isoenzymes. The activities of LDH1 and LDH2 isoenzymes were decreased in all applied doses of the complex 2, while the activity of LDH2 reduced using complex 1 in the same doses. Results of the present study suggest the neuro- and cardio protective potential of oral administration of complexes 1 and 2, as non-toxic compounds under physiological conditions. These protective effects are the result of their potent antioxidant activity.

scholarly journals Modulation of bladder function by luminal adenosine turnover and A1 receptor activation

2012 ◽  
Vol 303 (2) ◽  
pp. F279-F292 ◽  
Author(s):  
H. Sandeep Prakasam ◽  
Heather Herrington ◽  
James R. Roppolo ◽  
Edwin K. Jackson ◽  
Gerard Apodaca
Keyword(s):  
Receptor Activation ◽  
Adenosine Kinase ◽  
Nucleoside Transporters ◽  
Bladder Function ◽  
Extracellular Adenosine ◽  
Equilibrative Nucleoside Transporters ◽  
Lack Of Information ◽  
Bladder Activity ◽  
A1 Receptor ◽  
A Site

The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A1, A2A, A2B, and A3), and is a site of adenosine production. Although adenosine has a well-described protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed that the concentration of extracellular adenosine at the mucosal surface of the uroepithelium was regulated by ecto-adenosine deaminase and by equilibrative nucleoside transporters, whereas adenosine kinase and equilibrative nucleoside transporters modulated serosal levels. We further observed that enriching endogenous adenosine by blocking its routes of metabolism or direct activation of mucosal A1 receptors with 2-chloro- N6-cyclopentyladenosine (CCPA), a selective agonist, stimulated bladder activity by lowering the threshold pressure for voiding. Finally, CCPA did not quell bladder hyperactivity in animals with acute cyclophosphamide-induced cystitis but instead exacerbated their irritated bladder phenotype. In conclusion, we find that adenosine levels at both surfaces of the uroepithelium are modulated by turnover, that blocking these pathways or stimulating A1 receptors directly at the luminal surface promotes bladder contractions, and that adenosine further stimulates voiding in animals with cyclophosphamide-induced cystitis.

Curcumin protects rat heart mitochondria against anoxia–reoxygenation induced oxidative injury

2013 ◽  
Vol 91 (9) ◽  
pp. 715-723 ◽  
Author(s):  
PeiHan Xu ◽  
YaLi Yao ◽  
PengJun Guo ◽  
Ting Wang ◽  
BingWu Yang ◽  
...  
Keyword(s):  
Rat Heart ◽  
Respiratory Activity ◽  
Curcuma Longa ◽  
Ischemia Reperfusion ◽  
Respiratory Control ◽  
Protective Effects ◽  
Cyt C ◽  
Rat Heart Mitochondria ◽  
Nanomolar Range

It is an important therapeutic strategy to protect mitochondria from oxidative stress, especially during ischemia–reperfusion. Curcumin is a naturally occurring phenolic compound isolated as a yellow pigment from turmeric (Curcuma longa). This compound has received much attention due to its diversity of biological and pharmacological activities. In this study, an attempt has been made to evaluate the protective effects of curcumin on rat heart mitochondrial injuries induced by in vitro anoxia–reoxygenation. It was found that curcumin added before anoxia or immediately prior to reoxygenation exhibited remarkable protective effects against anoxia–reoxygenation induced oxidative damage to mitochondria, in concentrations ranging from picomoles to micromoles, with EC50s in the nanomolar range. The protective effects include inhibition of the decrease of state 3 respiratory activity, the decrease of respiratory control ratio (RCR) and ADP:oxygen (ADP:O) ratio, as well as the increase of state 4 respiratory activity; inhibition of the decrease of the membrane fluidity; inhibition of lipoperoxidation and protein carbonylation; as well as inhibition of the enhanced release of cardiolipin (CL) and cytochrome c (Cyt c). These results demonstrate the superior antioxidative properties of curcumin, and make it a promising candidate for the prevention and (or) therapy for ischemia–reperfusion injuries and the related free radical initiated diseases.

scholarly journals A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity

2002 ◽  
Vol 361 (2) ◽  
pp. 379-384 ◽  
Author(s):  
Richard GRAEFF ◽  
Hon Cheung LEE
Keyword(s):  
High Sensitivity ◽  
Physiological Conditions ◽  
Adp Ribosyl Cyclase ◽  
High Throughput Method ◽  
Plate Reader ◽  
Coupled Reactions ◽  
High Concentrations ◽  
Nanomolar Range ◽  
Fluorescence Plate Reader

cADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD+ that has now been established as a general Ca2+ messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.

scholarly journals Phosphorylation of adenosine in anoxic hepatocytes by an exchange reaction catalysed by adenosine kinase

1993 ◽  
Vol 290 (3) ◽  
pp. 679-684 ◽  
Author(s):  
F Bontemps ◽  
M Mimouni ◽  
G Van den Berghe
Keyword(s):  
Exchange Reaction ◽  
Rat Liver ◽  
High Speed ◽  
Kinase Activity ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Adenosine Kinase ◽  
Isolated Rat Hepatocytes ◽  
Total Absence ◽  
Adenylate Pool

The elevation of adenosine levels induced by anoxia in isolated rat hepatocytes has been shown to result mainly from an arrest of the recycling of the nucleoside by adenosine kinase [Bontemps, Vincent and Van den Berghe (1993) Biochem. J. 290, 671-677]. To assess the activity of the latter enzyme in intact hepatocytes, incorporation of radioactive adenosine into the cells' adenine nucleotides was measured. Unexpectedly, despite the near-absence of ATP in anoxic cells, 40% of 50 microM [8-14C]adenosine was still incorporated into adenylates over 5 min. Moreover, whereas unlabelled and labelled adenosine were utilized in parallel in normoxic cells, uptake of [8-14C]adenosine did not correspond to a net disappearance of adenosine in anoxic cells. Addition of 1 mM unlabelled adenosine to anoxic hepatocytes in which the adenine nucleotides had been prelabelled with [U-14C]adenine induced an immediate loss of their radioactivity. The latter was recovered in the form of adenosine, but the size of the adenylate pool was not modified. Taken together, these results suggest the occurrence of an exchange reaction between AMP and adenosine. Incubation of Sephadex G-25-filtered high-speed supernatants of rat liver with 20 microM [8-14C]adenosine, 10 mM MgCl2 and 1 mM AMP resulted in the labelling of AMP in the total absence of ATP. This labelling was influenced by effectors of both adenosine kinase and cytosolic IMP-GMP 5′-nucleotidase; the latter is known to catalyse an exchange reaction [Worku and Newby (1982) Biochem. J. 205, 503-510]. Chromatography of cytosolic fractions of rat liver on DEAE-Sepharose, followed by Sephacryl S-200 and AMP-Sepharose, demonstrated that the exchange reaction between adenosine and AMP co-purified with adenosine kinase. It is concluded that incorporation of labelled adenosine into adenine nucleotides should not be considered to be proof of adenosine kinase activity in anoxia.

scholarly journals NAD+ improves cognitive function and reduces neuroinflammation by ameliorating mitochondrial damage and decreasing ROS production in chronic cerebral hypoperfusion models through Sirt1/PGC-1α pathway

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Yao Zhao ◽  
Jiawei Zhang ◽  
Yaling Zheng ◽  
Yaxuan Zhang ◽  
Xiao Jie Zhang ◽  
...  
Keyword(s):  
Mitochondrial Damage ◽  
Chronic Cerebral Hypoperfusion ◽  
Cerebral Hypoperfusion ◽  
Ros Production ◽  
Protective Effects ◽  
Sprague Dawley ◽  
Mechanistic Pathway ◽  
Bv2 Microglia

Abstract Background Microglial-mediated neuroinflammation plays an important role in vascular dementia, and modulating neuroinflammation has emerged as a promising treatment target. Nicotinamide adenine dinucleotide (NAD+) shows anti-inflammatory and anti-oxidant effects in many neurodegenerative disease models, but its role in the chronic cerebral hypoperfusion (CCH) is still unclear. Methods The bilateral common carotid artery occlusion (BCCAO) was performed to establish CCH models in Sprague-Dawley rats. The rats were given daily intraperitoneal injection of NAD+ for 8 weeks. The behavioral test and markers for neuronal death and neuroinflammation were analyzed. Mitochondrial damage and ROS production in microglia were also assessed. RNA-seq was performed to investigate the mechanistic pathway changes. For in vitro studies, Sirt1 was overexpressed in BV2 microglial cells to compare with NAD+ treatment effects on mitochondrial injury and neuroinflammation. Results NAD+ administration rescued cognitive deficits and inhibited neuroinflammation by protecting mitochondria and decreasing ROS production in CCH rats. Results of mechanistic pathway analysis indicated that the detrimental effects of CCH might be associated with decreased gene expression of PPAR-γ co-activator1α (PGC-1α) and its upstream transcription factor Sirt1, while NAD+ treatment markedly reversed their decrease. In vitro study confirmed that NAD+ administration had protective effects on hypoxia-induced neuroinflammation and mitochondrial damage, as well as ROS production in BV2 microglia via Sirt1/PGC-1α pathway. Sirt1 overexpression mimicked the protective effects of NAD+ treatment in BV2 microglia. Conclusions NAD+ ameliorated cognitive impairment and dampened neuroinflammation in CCH models in vivo and in vitro, and these beneficial effects were associated with mitochondrial protection and ROS inhibition via activating Sirt1/PGC-1α pathway.

scholarly journals DL0410 Alleviates Memory Impairment in D-Galactose-Induced Aging Rats by Suppressing Neuroinflammation via the TLR4/MyD88/NF-κB Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-31
Author(s):  
Baoyue Zhang ◽  
Wenwen Lian ◽  
Jun Zhao ◽  
Zhe Wang ◽  
Ailin Liu ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Signaling Pathway ◽  
Oxidation Products ◽  
Inflammatory Factors ◽  
Morris Water Maze Test ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Aging Rats ◽  
Bv2 Cells ◽  
Bv2 Microglia

Oxidative stress and neuroinflammation have been demonstrated to be linked with Alzheimer’s disease (AD). In this study, we examined the protective effects of DL0410 in aging rats and explored the underlying mechanism against oxidative damage and neuroinflammation, which was then validated in LPS-stimulated BV2 microglia. We firstly investigated the improvement effects of DL0410 on learning and memory abilities and explored the potential mechanisms in D-gal-induced aging rats. An 8-week treatment with DL0410 significantly improved the learning and cognitive function of D-gal-stimulated Alzheimer’s-like rats in the Morris water maze test, step-down test, and novel object recognition test, and the therapeutic effect of DL0410 at 10 mg/kg was even better than that of donepezil. What is more, the results showed that DL0410 alleviated neuron injury, increased the number of synapses, and improved the level of postsynaptic density protein 95 (PSD95) in the hippocampus and cortex. Next, we examined the protective effects of DL0410 against oxidative damage and neuroinflammation. Our observations indicated that DL0410 reduced the production of harmful oxidation products and promoted the antioxidative system, decreased the levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), and increased anti-inflammatory cytokines IL-10. Moreover, DL0410 inhibited the activation of astrocytes and microglia and suppressed the activation of the TLR4/MyD88/NF-κB signaling pathway. The anti-inflammation effect of DL0410 was further confirmed in LPS-stimulated BV2 cells, and the results showed that DL0410 reduced the level of inflammatory factors and inhibited the activation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway in BV2 microglia. Molecular docking results indicated that DL0410 occupied the LPS recognition site in the TLR4/MD2 complex. Furthermore, the enhanced expression of claudin-1, claudin-5, occludin, CX43, and ZO-1 indicated that DL0410 protected the blood-brain barrier (BBB) integrity. Together, these results suggest that DL0410 exerts neuroprotective effects against hippocampus and cortex injury induced by D-galactose, and the possible mechanisms include antioxidative stress, antineuroinflammation, improving synaptic plasticity, and maintaining BBB integrity, which is mediated by the TLR4/MyD88/NF-κB signaling pathway inhibition. We suggest that DL0410 is a promising candidate for AD treatment.

scholarly journals Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons

2012 ◽  
Vol 107 (10) ◽  
pp. 2769-2781 ◽  
Author(s):  
J. M. Hawryluk ◽  
L. L. Ferrari ◽  
S. A. Keating ◽  
E. Arrigoni
Keyword(s):  
Basal Forebrain ◽  
Brain Slices ◽  
Cholinergic Neurons ◽  
Adenosine Kinase ◽  
Equilibrative Nucleoside Transporters ◽  
Basal Forebrain Cholinergic Neurons ◽  
Whole Cell Patch Clamp ◽  
Adenosine Uptake ◽  
Inhibitory Tone ◽  
Sleep Factor

Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A1 receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated.

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