- Nucleosides block AICAR-stimulated activation of AMPK in skeletal muscle and cancer cells. [Journal Article]
- AJAm J Physiol Cell Physiol 2018 Sep 19
- AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-aminoimidazole-4-carboxamide-1-β-D-rib...
AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an adenosine analogue, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as MEMα, contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B12, vitamin C and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.
- Physical exercise prevents alterations in purinergic system and oxidative status in lipopolysaccharide-induced sepsis in rats. [Journal Article]
- JCJ Cell Biochem 2018 Sep 19
- Sepsis is a generalized infection that involves alterations in inflammatory parameters, oxidant status, and purinergic signaling in many tissues. Physical exercise has emerged as a tool to prevent th...
Sepsis is a generalized infection that involves alterations in inflammatory parameters, oxidant status, and purinergic signaling in many tissues. Physical exercise has emerged as a tool to prevent this disease because of its anti-inflammatory and antioxidant properties. Thus, in this study, we investigated the effects of physical exercise on preventing alterations in purinergic system components, oxidative stress, and inflammatory parameters in lipopolysaccharide (LPS)-induced sepsis in rats. Male Wistar rats were divided into four groups: control, exercise (EX), LPS, and EX+LPS. The resisted physical exercise was performed for 12 weeks on a ladder with 1 m height. After 72 hours of the last exercise session, the animals received 2.5 mg/kg of LPS for induction of sepsis, and after 24 hours, lungs and blood samples were collected for analysis. The results showed that the exercise protocol used was able to prevent, in septic animals: (1) the increase in body temperature; (2) the increase of lipid peroxidation and reactive species levels in the lung, (3) the increase in adenosine triphosphate levels in serum; (4) the change in the activity of the enzymes ectonucleotidases in lymphocytes, partially; (5) the change in the density of purinergic enzymes and receptors in the lung, and (6) the increase of IL-6 and IL-1β gene expression. Our results revealed the involvement of purinergic signaling and oxidative damage in the mechanisms by which exercise prevents sepsis aggravations. Therefore, the regular practice of physical exercise is encouraged as a better way to prepare the body against sepsis complications.
- Demonstration of Pulmonary Vein Exit Block following Pulmonary Vein Isolation: A Novel Use for Adenosine. [Journal Article]
- JCJ Cardiovasc Electrophysiol 2018 Sep 19
- CONCLUSIONS: Adensoine administered during PV pacing allows transient visualization of local PV evoked potentials after PVI facilitating clearer demonstration of PV exit block in over 50% veins. This article is protected by copyright. All rights reserved.
- Both toxic and beneficial effects of pyocyanin contribute to the lifecycle of Pseudomonas aeruginosa. [Journal Article]
- MMMol Microbiol 2018 Sep 19
- Pseudomonas aeruginosa, an opportunistic pathogen, produces redox-active pigments called phenazines. Pyocyanin (PYO, the blue phenazine) plays an important role during biofilm development. Paradoxica...
Pseudomonas aeruginosa, an opportunistic pathogen, produces redox-active pigments called phenazines. Pyocyanin (PYO, the blue phenazine) plays an important role during biofilm development. Paradoxically, PYO auto-poisoning can stimulate cell death and release of extracellular DNA (eDNA), yet PYO can also promote survival within biofilms when cells are oxidant-limited. Here we identify the environmental and physiological conditions in planktonic culture that promote PYO-mediated cell death. We demonstrate that PYO auto-poisoning is enhanced when cells are starved for carbon. In the presence of PYO, cells activate a set of genes involved in energy-dependent defenses, including: (i) the oxidative stress response, (ii) RND efflux systems and (iii) iron-sulfur cluster biogenesis factors. P. aeruginosa can avoid PYO poisoning when reduced carbon is available, but blockage of adenosine triphosphate (ATP) synthesis either through carbon limitation or direct inhibition of the F0 F1 -ATP synthase triggers death and eDNA release. Finally, even though PYO is toxic to the majority of the population when cells are nutrient limited, a subset of cells is intrinsically PYO resistant. The effect of PYO on the producer population thus appears to be dynamic, playing dramatically different yet predictable roles throughout distinct stages of growth, helping rationalize its multifaceted contributions to biofilm development. This article is protected by copyright. All rights reserved.
- Generation of procoagulant collagen- and thrombin-activated platelets in platelet concentrates derived from buffy coat: the role of processing, pathogen inactivation, and storage. [Journal Article]
- TTransfusion 2018 Sep 19
- CONCLUSIONS: Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP.
- A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death. [Journal Article]
- JCJ Cell Physiol 2018 Sep 19
- Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage v...
Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn2+ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H2 O2 induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn2+ ]i , and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H2 O2 increased concentration-dependently the [Zn2+ ]i and caused lysosomal dysfunction and Zn2+ loss and, furthermore, mitochondrial Zn2+ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3',5,5'-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn2+ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn2+ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn2+ uptake in isolated mitochondria, which was prevented by TPEN. H2 O2 -induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H2 O2 -induced ROS generation, lysosomal dysfunction and Zn2+ release, and mitochondrial Zn2+ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn2+ release, and mitochondrial Zn2+ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.
- Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence. [Journal Article]
- CEClin Exp Immunol 2018 Sep 19
- Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(-) exosomes by immune capt...
Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(-) exosomes by immune capture. CD3(-) exosomes were largely tumour-derived (CD44v3+ ). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3(+) or CD3(-) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4+ CD39+ regulatory T cells (Treg ) co-incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3(-) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5'-AMP and purines was significantly higher in Treg co-incubated with CD3(-) than CD3(+) exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3(+) than CD3(-) exosomes. ADA and CD26 levels in CD3(+) exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3(+) exosomes with no increase in CD3(-) exosomes. The opposite roles of CD3(+) ADA+ CD26+ and CD3(-) CD44v3+ adenosine-producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.
- Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation. [Journal Article]
- EMEndocrinol Metab (Seoul) 2018; 33(3):403-412
- CONCLUSIONS: Altogether, these data suggest that S1P is an essential factor in the maintenance of β-cell function and survival through its regulation of mitochondrial action and PHB expression.
- ATP mediates a negative autocrine signal on stimulus-secretion coupling in mouse pancreatic β-cells. [Journal Article]
- EEndocrine 2018 Sep 18
- CONCLUSIONS: It is suggested that extracellular ATP which is co-secreted with insulin in a pulsatile manner during glucose-stimulated exocytosis provides a negative feedback signal driving β-cell oscillations in co-operation with Ca2+ and other signals.
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- Effect of Adenosine and Adenosine Receptor Antagonists on Retinal Müller Cell Inwardly Rectifying Potassium Channels under Exogenous Glutamate Stimulation. [Journal Article]
- BRBiomed Res Int 2018; 2018:2749257
- The vitreousness of glaucoma subjects contains elevated glutamate, and excessive extracellular glutamate is toxic to retinal neurons. Therefore, glutamate clearance is potentially impaired in the ret...
The vitreousness of glaucoma subjects contains elevated glutamate, and excessive extracellular glutamate is toxic to retinal neurons. Therefore, glutamate clearance is potentially impaired in the retina of glaucoma subjects. Müller cells play an important role in maintaining low extracellular levels of neurotransmitters, such as glutamate. A better understanding of the cross-talk between adenosine and glutamate may provide a better characterization of the regulatory network in Müller cells. Here, Müller cells were purified from the rat retina on postnatal day 5 using the papain digestion method. Application of increasing concentrations of glutamate (0-20 mmol/L) caused a dose-dependent decrease in the expression levels of Kir4.1, Kir2.1, GLAST, and GS. Exogenous adenosine regulated Kir channels and subsequently promoted GLAST and GS expression levels in Müller cells under exogenous glutamate stimulation. These effects were partly dependent on adenosine receptors.