A novel adenosine precursor 2',3'-cyclic adenosine monophosphate inhibits formation of post-surgical adhesions.

PubMed

Forman, Mervyn B; Gillespie, Delbert G; Cheng, Dongmei; Jackson, Edwin K

2014-09-01

Intraperitoneal adenosine reduces abdominal adhesions. However, because of the ultra-short half-life and low solubility of adenosine, optimal efficacy requires multiple dosing. Here, we compared the ability of potential adenosine prodrugs to inhibit post-surgical abdominal adhesions after a single intraperitoneal dose. Abdominal adhesions were induced in mice using an electric toothbrush to damage the cecum. Also, 20 μL of 95 % ethanol was applied to the cecum to cause chemically induced injury. After injury, mice received intraperitoneally either saline (n = 18) or near-solubility limit of adenosine (23 mmol/L; n = 12); 5'-adenosine monophosphate (75 mmol/L; n = 11); 3'-adenosine monophosphate (75 mmol/L; n = 12); 2'-adenosine monophosphate (75 mmol/L; n = 12); 3',5'-cyclic adenosine monophosphate (75 mmol/L; n = 19); or 2',3'-cyclic adenosine monophosphate (75 mmol/L; n = 20). After 2 weeks, adhesion formation was scored by an observer blinded to the treatments. In a second study, intraperitoneal adenosine levels were measured using tandem mass spectrometry for 3 h after instillation of 2',3'-cyclic adenosine monophosphate (75 mmol/L) into the abdomen. The order of efficacy for attenuating adhesion formation was: 2',3'-cyclic adenosine monophosphate > 3',5'-cyclic adenosine monophosphate ≈ adenosine > 5'-adenosine monophosphate ≈ 3'-adenosine monophosphate ≈ 2'-adenosine monophosphate. The groups were compared using a one-factor analysis of variance, and the overall p value for differences between groups was p < 0.000001. Intraperitoneal administration of 2',3'-cAMP yielded pharmacologically relevant levels of adenosine in the abdominal cavity for >3 h. Administration of 2',3'-cyclic adenosine monophosphate into the surgical field is a unique, convenient and effective method of preventing post-surgical adhesions by acting as an adenosine prodrug.

Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase.

PubMed

Rhim, Ji-Heon; Jang, Ik-Soon; Song, Kye-Yong; Ha, Moon-Kyung; Cho, Sung-Chun; Yeo, Eui-Ju; Park, Sang Chul

2008-08-01

This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation.

PubMed

Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua

2013-01-01

Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

PubMed Central

Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

2008-01-01

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

PubMed

Cassera, María B; Hazleton, Keith Z; Riegelhaupt, Paul M; Merino, Emilio F; Luo, Minkui; Akabas, Myles H; Schramm, Vern L

2008-11-21

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Methylene blue induces macroautophagy through 5′ adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation

PubMed Central

Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua

2013-01-01

Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5′ adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling. PMID:23653592

5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

PubMed

Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

2016-12-01

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections. © 2016 John Wiley & Sons Ltd.

Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

PubMed

Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

2015-03-25

This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

Donor pretreatment with adenosine monophosphate-activated protein kinase activator protects cardiac grafts from cold ischaemia/reperfusion injury.

PubMed

Yang, Chao; Xu, Honglai; Cai, Lanjun; Du, Xiaoxiao; Jiang, Yinan; Zhang, Yong; Zhou, Hongmin; Chen, Zhonghua Klaus

2016-05-01

Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy metabolism and has been shown to be protective in ischaemia/reperfusion injury (IRI). We hypothesized that preactivation of AMPK with an activator before donor heart procurement could protect heart grafts from cold IRI. Donor Sprague-Dawley rats were injected intravenously with AMPK activator 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) or vehicle 30 min before heart procurement. Heart grafts were then preserved in histidine-tryptophan-ketoglutarate (HTK) solution at 4°C for 8 h. After preservation, grafts were immediately mounted on the Langendorff perfusion system and perfused with Krebs-Henseleit buffer at 37°C for 1 h. Adenosine triphosphate (ATP) and malondialdehyde (MDA) content in graft tissue were quantified post-preservation and post-reperfusion. After reperfusion, isolated heart function was assessed using a pressure transducer; cumulative release of creatine kinase (CK) and lactate dehydrogenase (LDH) into the perfusate was measured to assess cardiomyocyte necrosis; ultrastructural changes in the mitochondria of the grafts were examined using transmission electron microscopy (TEM). After preservation, myocardial ATP content in the pretreated hearts was significantly higher than in the control hearts (3.247 ± 0.3034 vs 1.817 ± 0.2533 µmol/g protein; P < 0.05). AICAR-pretreated heart grafts exhibited significantly higher coronary flow (9.667 ± 0.3159 vs 8.033 ± 0.2459 ml/min; P < 0.05) and left ventricular developing pressure (58.67 ± 2.894 vs 42.67 ± 3.333 mmHg; P < 0.05) than the vehicle treated after reperfusion. Cumulative release of CK (300.0 ± 25.30 vs 431.7 ± 42.39 U/l; P < 0.05) and LDH (228.0 ± 16.68 vs 366.8 ± 57.41 U/l; P < 0.05) in the perfusate was significantly lower in the AICAR-pretreated group than that in the control group. Myocardial MDA content was also reduced in the pretreated group (0.5167 ± 0.1046 vs 0.9333 ± 0.1333 nmol

Genistein promotes insulin action through adenosine monophosphate-activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet.

PubMed

Arunkumar, Elumalai; Anuradha, Carani Venkatraman

2012-08-01

Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of activation of 5'-adenosine monophosphate-activated protein kinase in gastric ulcer healing in diabetic rats.

PubMed

Baraka, Azza M; Deif, Maha M

2011-01-01

The potential utility of 5'-adenosine monophosphate-activated protein kinase (AMPK)-activating agents, such as metformin, in inducing angiogenesis, could be a promising approach to promote healing of gastric ulcers complicated by diabetes mellitus. The aim of the present study was to assess the effect of a drug that activates AMPK, namely metformin, in gastric ulcer healing in streptozotocin-induced diabetic rats. Forty male Wistar albino rats were made diabetic by intraperitoneal (i.p.) streptozotocin injection and 10 rats were injected i.p. by a single dose of physiological saline. Six weeks following streptozotocin or saline injection, gastric ulcers were induced by serosal application of acetic acid. Three days after acetic acid application, rats were divided into group 1 (nondiabetic control), group 2 (streptozotocin-injected rats), groups 3-5 (streptozotocin-injected rats treated with metformin or metformin and an inhibitor of AMPK, namely compound C or pioglitazone) for 7 days following acetic acid application. Administration of metformin, but not pioglitazone, resulted in a significant decrease in the gastric ulcer area, a significant increase in epithelial regeneration assessed histologically, a significant increase in the number of microvessels in the ulcer margin, a significant increase in gastric vascular endothelial growth factor concentration and gastric von Willebrand factor as well as a significant increase in gastric phospho-AMPK. Compound C, an inhibitor of AMPK, blocked metformin-induced changes in assessed parameters suggesting that the effect of metformin was mediated mainly through activation of AMPK. Our results suggest the feasibility of a novel treatment strategy, namely drugs activating AMPK, for patients in whom impairment of ulcer healing constitutes a secondary complication of diabetes mellitus. Copyright © 2011 S. Karger AG, Basel.

Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

PubMed

Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

2011-06-01

The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

Prostacyclin regulates spinal nociceptive processing through cyclic adenosine monophosphate-induced translocation of glutamate receptors.

PubMed

Schuh, Claus Dieter; Brenneis, Christian; Zhang, Dong Dong; Angioni, Carlo; Schreiber, Yannick; Ferreiros-Bouzas, Nerea; Pierre, Sandra; Henke, Marina; Linke, Bona; Nüsing, Rolf; Scholich, Klaus; Geisslinger, Gerd

2014-02-01

Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

PubMed

Chen, Baolin; Wu, Qiang; Xiong, Zhaojun; Ma, Yuedong; Yu, Sha; Chen, Dandan; Huang, Shengwen; Dong, Yugang

2016-09-01

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

GROWTH AND DEVELOPMENT SYMPOSIUM: Adenosine monophosphate-activated protein kinase and mitochondria in Rendement Napole pig growth.

PubMed

Scheffler, T L; Gerrard, D E

2016-09-01

The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth.

Retinoic acid receptor-related orphan receptor α-induced activation of adenosine monophosphate-activated protein kinase results in attenuation of hepatic steatosis.

PubMed

Kim, Eun-Jin; Yoon, Young-Sil; Hong, Suckchang; Son, Ho-Young; Na, Tae-Young; Lee, Min-Ho; Kang, Hyun-Jin; Park, Jinyoung; Cho, Won-Jea; Kim, Sang-Gun; Koo, Seung-Hoi; Park, Hyeung-geun; Lee, Mi-Ock

2012-05-01

There is increasing evidence that the retinoic acid receptor-related orphan receptor α (RORα) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORα in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORα attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor α (LXRα). First, RORα and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORα, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRα and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORα interacted physically with LXRα and inhibited the LXRα response element in the promoter of LXRα, indicating that RORα interrupts the autoregulatory activation loop of LXRα. Third, infection with adenovirus encoding RORα suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORα protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORα via tail-vein injection of adenovirus (Ad)-RORα decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORα, thereby inducing activation of AMPK and repression of LXRα. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. We found that RORα induced activation of AMPK and inhibition of the lipogenic function of LXRα, which may be key phenomena that provide the beneficial effects of RORα against hepatic steatosis

Effects of tiletamine on the adenosine monophosphate-activated protein kinase signaling pathway in the rat central nervous system.

PubMed

Su, Li-Xue; Shi, Xing-Xing; Yang, Peng; Chen, Hao; Li, Xin; Fan, Hong-Gang; Wang, Hong-Bin

2017-10-01

The dissociative anesthetic tiletamine, which acts on the central nervous system (CNS), is widely used in veterinary medicine and animal experiments. Recent studies indicate that adenosine 5'-monophosphate activated protein kinase (AMPK) plays a key role in the analgesic action of tiletamine. In the present study, the effects of tiletamine on the AMPK signaling pathway in rats were investigated. Sprague-Dawley rats were injected intraperitoneally with tiletamine and executed at 10, 20, 40 and 60min post injection. The cerebral cortex, hippocampus, thalamus, cerebellum and brainstem were immediately taken out to evaluate the mRNA and protein phosphorylation levels of liver kinase B1 (LKB1), AMPKα and eIF4E-binding protein 1 (4EBP1) using quantitative real-time polymerase chain reaction and western blot analysis. Tiletamine increased AMPK mRNA expression in the rat brain (P<0.01). Increased mRNA expression of AMPK was accompanied by an increase in phosphorylation of LKB1, resulting in significant decreases in the phosphorylation levels of 4EBP1 in the corresponding brain regions (P<0.01). In summary, the findings indicate that tiletamine regulates the mRNA expression and protein phosphorylation levels of LKB1, AMPK and 4EBP1 in the CNS, suggesting that the analgesic effect of the anesthetic is mediated, at least in part, by the AMPK signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Time-resolved photoelectron spectroscopy of adenosine and adenosine monophosphate photodeactivation dynamics in water microjets

NASA Astrophysics Data System (ADS)

Williams, Holly L.; Erickson, Blake A.; Neumark, Daniel M.

2018-05-01

The excited state relaxation dynamics of adenosine and adenosine monophosphate were studied at multiple excitation energies using femtosecond time-resolved photoelectron spectroscopy in a liquid water microjet. At pump energies of 4.69-4.97 eV, the lowest ππ* excited state, S1, was accessed and its decay dynamics were probed via ionization at 6.20 eV. By reversing the role of the pump and probe lasers, a higher-lying ππ* state was excited at 6.20 eV and its time-evolving photoelectron spectrum was monitored at probe energies of 4.69-4.97 eV. The S1 ππ* excited state was found to decay with a lifetime ranging from ˜210 to 250 fs in adenosine and ˜220 to 250 fs in adenosine monophosphate. This lifetime drops with increasing pump photon energy. Signal from the higher-lying ππ* excited state decayed on a time scale of ˜320 fs and was measureable only in adenosine monophosphate.

"Preconditioning" with latrepirdine, an adenosine 5'-monophosphate-activated protein kinase activator, delays amyotrophic lateral sclerosis progression in SOD1(G93A) mice.

PubMed

Coughlan, Karen S; Mitchem, Mollie R; Hogg, Marion C; Prehn, Jochen H M

2015-02-01

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a master regulator of energy balance. As energy imbalance is documented as a key pathologic feature of amyotrophic lateral sclerosis (ALS), we investigated AMPK as a pharmacologic target in SOD1(G93A) mice. We noted a strong activation of AMPK in lumbar spinal cords of SOD1(G93A) mice. Pharmacologic activation of AMPK has shown protective effects in neuronal "preconditioning" models. We tested the hypothesis that "preconditioning" with a small molecule activator of AMPK, latrepirdine, exerts beneficial effects on disease progression. SOD1(G93A) mice (n = 24 animals per group; sex and litter matched) were treated with latrepirdine (1 μg/kg, intraperitoneal) or vehicle from postnatal day 70 to 120. Treatment with latrepirdine increased AMPK activity in primary mouse motor neuron cultures and in SOD1(G93A) lumbar spinal cords. Mice "preconditioned" with latrepirdine showed a delayed symptom onset and a significant increase in life span (p < 0.01). Our study suggests that "preconditioning" with latrepirdine may represent a possible therapeutic strategy for individuals harboring ALS-associated gene mutations who are at risk for developing ALS. Copyright © 2015 Elsevier Inc. All rights reserved.

Direct Activation of Adenosine Monophosphate-Activated Protein Kinase (AMPK) by PF-06409577 Inhibits Flavivirus Infection through Modification of Host-Cell Lipid Metabolism.

PubMed

Jiménez de Oya, Nereida; Blázquez, Ana-Belén; Casas, Josefina; Saiz, Juan-Carlos; Martín Acebes, Miguel A

2018-04-30

Mosquito-borne flaviviruses are a group of RNA viruses that constitute global threats for human and animal health. Replication of these pathogens is strictly dependent on cellular lipid metabolism. We have evaluated the effect of the pharmacological activation of Adenosine Monophosphate-activated Protein Kinase (AMPK), a master regulator of lipid metabolism, on the infection of three medically relevant flaviviruses: West Nile virus (WNV), Zika virus (ZIKV) and dengue virus (DENV). WNV is responsible for recurrent outbreaks of meningitis and encephalitis affecting humans and horses worldwide. ZIKV has caused a recent pandemic associated with birth defects (microcephaly), reproductive disorders, and severe neurological complications (Guillain-Barré syndrome). DENV is the etiological agent of the most prevalent mosquito-borne viral disease that can induce a potentially lethal complication called severe dengue. Our results showed, for the first time, that activation of AMPK using the specific small molecule activator PF-06409577 reduced both WNV, ZIKV, and DENV infection. This antiviral effect was associated to an impairment of viral replication due to the modulation of host cell lipid metabolism exerted by the compound. These results support that the pharmacological activation of AMPK, which currently constitutes an important pharmacological target for human diseases, could also provide a feasible approach for broad-spectrum host-directed antiviral discovery. Copyright © 2018 American Society for Microbiology.

Adenosine Kinase: Exploitation for Therapeutic Gain

PubMed Central

2013-01-01

Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

PubMed

Hsu, Chao-Yu; Lin, Chun-Hsiang; Lin, Jiun-Tsai; Cheng, Yi-Fang; Chen, Han-Min; Kao, Shao-Hsuan

2015-09-01

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI‑F706 on the human renal carcinoma cell line 786‑O and the underlying mechanisms. The results revealed that ENERGI‑F706 (0.2‑0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786‑O cells, ENERGI‑F706 exerted less suppressive effects on the viability of the human non‑tumorigenic renal cell line HK‑2. Flow cytometric analysis showed that ENERGI‑F706 contributed to cell cycle arrest at S‑phase and triggered apoptosis of 786‑O cells. Immunoblot analysis revealed that anti‑apoptotic B‑cell lymphoma 2 (Bcl‑2) levels were reduced and pro‑apoptotic Bcl‑2‑associated X protein levels were diminished. In addition, activation of caspase‑9, caspase‑3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786‑O cells in response to ENERGI‑F706. Effects of ENERGI‑F706 on AMPK cascades were investigated and the results showed that ENERGI‑F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl‑2, cleavage of caspase‑3 and PARP as well as suppressed cell viability induced by ENERGI‑F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI‑F706 significantly suppressed the viability of 786‑O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI‑F706 may be suitable for the treatment of renal cell carcinoma.

The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy

PubMed Central

2012-01-01

Introduction Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown. Methods Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR. Results Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ. Conclusions These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.

PubMed

Kivi, Rait; Solovjova, Karina; Haljasorg, Tõiv; Arukuusk, Piret; Järv, Jaak

2016-12-01

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase catalytic subunit.

PubMed

Akamine, Pearl; Madhusudan; Brunton, Laurence L; Ou, Horng D; Canaves, Jaume M; Xuong, Nguyen-huu; Taylor, Susan S

2004-01-13

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selective-characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3',5'-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium- and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.

Genetics Home Reference: adenosine monophosphate deaminase deficiency

MedlinePlus

... view the expand/collapse boxes. Description Adenosine monophosphate (AMP) deaminase deficiency is a condition that can affect ... for movement ( skeletal muscles ). In many affected individuals, AMP deaminase deficiency does not cause any symptoms. People ...

Estrogen regulates energy metabolic pathway and upstream adenosine 5'-monophosphate-activated protein kinase and phosphatase enzyme expression in dorsal vagal complex metabolosensory neurons during glucostasis and hypoglycemia.

PubMed

Tamrakar, Pratistha; Ibrahim, Baher A; Gujar, Amit D; Briski, Karen P

2015-02-01

The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-β in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury. © 2014 Wiley Periodicals, Inc.

Adenosine monophosphate-activated kinase, AMPK, is involved in the maintenance of the quality of extended boar semen during long-term storage.

PubMed

Martin-Hidalgo, David; Hurtado de Llera, Ana; Yeste, Marc; Cruz Gil, M; Bragado, M Julia; Garcia-Marin, Luis J

2013-09-01

Boar semen preservation for later use in artificial insemination is performed by diluting semen in an appropriate medium and then lowering the temperature to decrease spermatozoa metabolism. The adenosine monophosphate-activated kinase, AMPK, is a key cell energy sensor that controls cell metabolism and recently has been identified in boar spermatozoa. Our aim was to investigate the role of AMPK in spermatozoa functional parameters including motility, mitochondrial membrane potential, plasma membrane integrity, acrosome integrity, and cell viability during long-term boar semen storage at 17 °C in Beltsville thawing solution. Boar seminal doses were diluted in Beltsville thawing solution in the presence or absence of different concentrations of AMPK inhibitor, compound C (1, 10, and 30 μM) and evaluations were performed at 1, 2, 4, 7, or 10 days. Data demonstrate that AMPK becomes phosphorylated at threonine(172) (active) during storage of boar semen reaching maximum levels at Day 7. Moreover, AMPK inhibition during boar semen storage causes: (1) a potent inhibition of spermatozoa motility; (2) a reduction in the percentage of spermatozoa showing high mitochondria membrane potential; (3) a rise in the percentage of spermatozoa displaying high plasma membrane scrambling; and (4) a loss of acrosomal membrane integrity. Our study suggests that AMPK activity plays an important role in the maintenance of the spermatozoa quality during long-term storage of boar semen. Copyright © 2013 Elsevier Inc. All rights reserved.

Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

PubMed

Ido, Yasuo

2016-07-01

Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. © 2016 The Author. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Ginsenoside Compound K suppresses the hepatic gluconeogenesis via activating adenosine-5'monophosphate kinase: A study in vitro and in vivo.

PubMed

Wei, Shengnan; Li, Wei; Yu, Yang; Yao, Fan; A, Lixiang; Lan, Xiaoxin; Guan, Fengying; Zhang, Ming; Chen, Li

2015-10-15

Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenoside. We have reported that CK presented anti-diabetic effect via diminishing the expressions of hepatic gluconeogenesis key enzyme. Here, we further explore the possible mechanism of CK on suppression hepatic gluconeogenesis via activation of adenosine-5'monophosphate kinase (AMPK) on type 2 diabetes mice in vivo and in HepG2 cells. Type 2 diabetes mice model was developed by high fat diet combined with STZ injection. 30mg/kg/d CK was orally administrated for 4weeks, the fasting blood glucose level and 2h OGTT were conducted, and the protein expression of AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase) were examined. The mechanism of Compound K on hepatic gluconeogenesis was further explored in HepG2 hepatocytes. Glucose production, the protein expression of AMPK, PEPCK, G6pase and PGC-1α, hepatic nuclear factor 4α (HNF-4α) and forkhead transcription factor O1 (FOXO1) were determined after Compound K treatment at the presence of AMPK inhibitor Compound C. We observed that CK inhibited the expression of PEPCK and G6Pase in the liver and in HepG2 hepatocytes. Meanwhile, CK treatment remarkably increased the activation of AMPK, while decreasing the expressions of PGC-1α, HNF-4α and FOXO1. However, AMPK inhibitor Compound C could reverse these effects of CK on gluconeogenesis in part. The results indicated that the effect of CK on suppression hepatic gluconeogenesis might be via the activation the AMPK activity. Copyright © 2015. Published by Elsevier Inc.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

PubMed

Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

2015-12-15

Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. Copyright © 2015 Elsevier B.V. All rights reserved.

Adenosine monophosphate-activated protein kinase is required for pulmonary artery smooth muscle cell survival and the development of hypoxic pulmonary hypertension.

PubMed

Ibe, Joyce Christina F; Zhou, Qiyuan; Chen, Tianji; Tang, Haiyang; Yuan, Jason X-J; Raj, J Usha; Zhou, Guofei

2013-10-01

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate-activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension.

SIRT1/Adenosine Monophosphate-Activated Protein Kinase α Signaling Enhances Macrophage Polarization to an Anti-inflammatory Phenotype in Rheumatoid Arthritis

PubMed Central

Park, So Youn; Lee, Sung Won; Lee, Sang Yeob; Hong, Ki Whan; Bae, Sun Sik; Kim, Koanhoi; Kim, Chi Dae

2017-01-01

Macrophages are crucially involved in the pathogenesis of rheumatoid arthritis (RA). Macrophages of the M1 phenotype act as pro-inflammatory mediators in synovium, whereas those of the M2 phenotype suppress inflammation and promote tissue repair. SIRT1 is a class 3 histone deacetylase with anti-inflammatory characteristics. However, the role played by SIRT1 in macrophage polarization has not been defined in RA. We investigated whether SIRT1 exerts anti-inflammatory effects by modulating M1/M2 polarization in macrophages from RA patients. In this study, SIRT1 activation promoted the phosphorylation of an adenosine monophosphate-activated protein kinase (AMPK) α/acetyl-CoA carboxylase in macrophages exposed to interleukin (IL)-4, and that this resulted in the expressions of M2 genes, including MDC, FcεRII, MrC1, and IL-10, at high levels. Furthermore, these expressions were inhibited by sirtinol (an inhibitor of SIRT1) and compound C (an inhibitor of AMPK). Moreover, SIRT1 activation downregulated LPS/interferon γ-mediated NF-κB activity by inhibiting p65 acetylation and the expression of M1 genes, such as CCL2, iNOS, IL-12 p35, and IL-12 p40. Macrophages from SIRT1 transgenic (Tg)-mice exhibited enhanced polarization of M2 phenotype macrophages and reduced polarization of M1 phenotype macrophages. In line with these observations, SIRT1-Tg mice showed less histological signs of arthritis, that is, lower TNFα and IL-1β expressions and less severe arthritis in the knee joints, compared to wild-type mice. Taken together, the study shows activation of SIRT1/AMPKα signaling exerts anti-inflammatory activities by regulating M1/M2 polarization, and thereby reduces inflammatory responses in RA. Furthermore, it suggests that SIRT1 signaling be viewed as a therapeutic target in RA. PMID:28966618

Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation.

PubMed

Fuentes, E; Badimon, L; Caballero, J; Padró, T; Vilahur, G; Alarcón, M; Pérez, P; Palomo, I

2014-03-03

Platelet activation is relevant to a variety of acute thrombotic events. We sought to examine adenosine 5'-monophosphate (AMP) mechanisms of action in preventing platelet activation, thrombus formation and platelet-related inflammatory response. We assessed the effect of AMP on 1) P-selectin expression and GPIIb/IIIa activation by flow cytometry; 2) Platelet aggregation and ATP secretion induced by ADP, collagen, TRAP-6, convulxin and thrombin; 3) Platelet rolling and firm adhesion, and platelet-leukocyte interactions under flow-controlled conditions; and, 4) Platelet cAMP levels, sP-selectin, sCD40L, IL-1β, TGF-β1 and CCL5 release, PDE3A activity and PKA phosphorylation. The effect of AMP on in vivo thrombus formation was also evaluated in a murine model. The AMP docking with respect to A2 adenosine receptor was determined by homology. AMP concentration-dependently (0.1 to 3 mmol/l) inhibited P-selectin expression and GPIIb/IIIa activation, platelet secretion and aggregation induced by ADP, collagen, TRAP-6 and convulxin, and diminished platelet rolling and firm adhesion. Furthermore, AMP induced a marked increase in the rolling speed of leukocytes retained on the platelet surface. At these concentrations AMP significantly decreased inflammatory mediator from platelet, increased intraplatelet cAMP levels and inhibited PDE3A activity. Interestingly, SQ22536, ZM241385 and SCH58261 attenuated the antiplatelet effect of AMP. Docking experiments revealed that AMP had the same orientation that adenosine inside the A2 adenosine receptor binding pocket. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. Considering the successful use of combined antiplatelet therapy, AMP may be further developed as a novel antiplatelet agent.

Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

PubMed

Li, Kai; Zhang, Haipeng; Qiu, Jianguang; Lin, Yuan; Liang, Jiankai; Xiao, Xiao; Fu, Liwu; Wang, Fang; Cai, Jing; Tan, Yaqian; Zhu, Wenbo; Yin, Wei; Lu, Bingzheng; Xing, Fan; Tang, Lipeng; Yan, Min; Mai, Jialuo; Li, Yuan; Chen, Wenli; Qiu, Pengxin; Su, Xingwen; Gao, Guangping; Tai, Phillip W L; Hu, Jun; Yan, Guangmei

2016-02-01

Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy.

Isolated adrenal cells: adrenocorticotropic hormone, calcium, steroidogenesis, and cyclic adenosine monophosphate.

PubMed

Sayers, G; Beall, R J; Seelig, S

1972-03-10

Corticosterone production by isolated adrenal cells in response to adrenocorticotropic hormone is reduced when the cells are incubated in a medium that contains no calcium. This reduction is associated with an equal reduction of accumulation of cyclic adenosine monophosphate. Production of corticosterone and accumulation of cyclic adenosine monophosphate are increased when the calcium concentration in the medium is increased (from zero to 7.65 millimolar). This is in contrast to the situation in "subcellular membrane fragments" of adrenal tissue where high calcium in the medium (> 1.0 millimolar) inhibits cyclic adenosine monophosphate accumulation. We propose that adenyl cyclase in the intact plasma membrane is located in a compartment wherein calcium concentration is low and remains unaffected by the concentration of calcium in the extracellular space. It is proposed that, as the concentration of calcium in the incubation medium is increased from zero to 7.65 millimolar, the strength of the signal generated by the interaction of adrenocorticotropic hormone with its receptor and transmitted to the adenyl cyclase compartment is proportionately increased.

Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

DOEpatents

Croteau, Rodney B.; Lange, Bernd M.

2001-01-01

A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

PubMed

Yang, Ya-Chun; Wang, Yen-Ting; Tseng, Wei-Lung

2017-03-22

Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe 3 O 4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe 3 O 4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H 2 O 2 ) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe 3 O 4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe 2+ /Fe 3+ sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H 2 O 2 . Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe 3 O 4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe 3 O 4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe 3 O 4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe 3 O 4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe 3 O 4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

PubMed

Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

2015-09-01

A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver. Copyright © 2015 Elsevier Inc. All rights reserved.

Photo protection of RNA building blocks: Adenosine 5‧-monophosphate, cytidine 5‧-monophosphate and cytosine

NASA Astrophysics Data System (ADS)

Nielsen, Jakob Brun; Thøgersen, Jan; Jensen, Svend Knak; Keiding, Søren Rud

2013-04-01

Photoprotection of the RNA nucleotides adenosine 5'-monophosphate and cytidine 5'-monophosphate, and the nucleobase cytosine was studied using UV pump, IR probe femtosecond transient absorption spectroscopy. The excitation energy is contained in the aromatic ring system, protecting the RNA backbone. All three molecules dissipate the excitation energy by internal conversion and subsequent vibrational relaxation to the electronic ground state in less than 10 ps. In addition, a second deactivation channel is found in cytidine 5'-monophosphate, illustrated by a signal at 1563 cm-1 with a lifetime of 33 ps assigned to an nπ∗ state in agreement with observations in the UV region.

Neutrophil-derived 5′-Adenosine Monophosphate Promotes Endothelial Barrier Function via CD73-mediated Conversion to Adenosine and Endothelial A2B Receptor Activation

PubMed Central

Lennon, Paul F.; Taylor, Cormac T.; Stahl, Gregory L.; Colgan, Sean P.

1998-01-01

During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration. PMID:9782120

Mulberry leaf polyphenol extract induced apoptosis involving regulation of adenosine monophosphate-activated protein kinase/fatty acid synthase in a p53-negative hepatocellular carcinoma cell.

PubMed

Yang, Tzi-Peng; Lee, Huei-Jane; Ou, Ting-Tsz; Chang, Ya-Ju; Wang, Chau-Jong

2012-07-11

The polyphenols in mulberry leaf possess the ability to inhibit cell proliferation, invasion, and metastasis of tumors. It was reported that the p53 status plays an important role in switching apoptosis and the cell cycle following adenosine monophosphate-activated protein kinase (AMPK) activation. In this study, we aimed to detect the effect of the mulberry leaf polyphenol extract (MLPE) on inducing cell death in p53-negative (Hep3B) and p53-positive (Hep3B with transfected p53) hepatocellular carcinoma cells and also to clarify the role of p53 in MLPE-treated cells. After treatment of the Hep3B cells with MLPE, apoptosis was induced via the AMPK/PI3K/Akt and Bcl-2 family pathways. Transient transfection of p53 into Hep3B cells led to switching autophagy instead of apoptosis by MLPE treatment. We demonstrated that acridine orange staining and protein expressions of LC-3 and beclin-1 were increased in p53-transfected cells. These results implied induction of apoptosis or autophagy in MLPE-treated hepatocellular carcinoma cells can be due to the p53 status. We also found MLPE can not only activate AMPK but also diminish fatty acid synthase, a molecular target for cancer inhibition. At present, our results indicate MLPE can play an active role in mediating the cell death of hepatocellular carcinoma cells and the p53 might play an important role in regulating the death mechanisms.

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

PubMed

Bilodeau-Goeseels, Sylvie

2011-01-01

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

6-Gingerol modulates proinflammatory responses in dextran sodium sulfate (DSS)-treated Caco-2 cells and experimental colitis in mice through adenosine monophosphate-activated protein kinase (AMPK) activation.

PubMed

Chang, Kuei-Wen; Kuo, Cheng-Yi

2015-10-01

6-gingerol has been reported to have anti-inflammatory effects in different experimental settings. The present study aimed at evaluating the effect of 6-gingerol on dextran sodium sulfate (DSS)-induced barrier impairment and inflammation in vitro and in vivo. a differentiated Caco-2 monolayer was exposed to DSS and treated with different concentrations of 6-gingerol (0, 1, 5, 10, 50, and 100 μM). Changes in intestinal barrier function were determined using transepithelial electrical resistance (TEER). The anti-inflammatory activity of 6-gingerol was examined as changes in the expression of proinflammatory cytokine using quantitative real-time PCR. Western blotting was employed to determine the activation of adenosine monophosphate-activated protein kinase (AMPK). Mice with DSS-induced colitis were given different oral dosages of 6-gingerol daily for 14 days. Body weight and colon inflammation were evaluated, and level of proinflammatory cytokines in colon tissues was measured. 6-gingerol treatment was shown to restore impaired intestinal barrier function and to suppress proinflammatory responses in DSS-treated Caco-2 monolayers. We found that AMPK was activated on 6-gingerol treatment in vitro. In animal studies, 6-gingerol significantly ameliorated DSS-induced colitis by restoration of body weight loss, reduction in intestinal bleeding, and prevention of colon length shortening. In addition, 6-gingerol suppressed DSS-elevated production of proinflammatory cytokines (IL-1β, TNFα, and IL-12). our findings highlight the protective effects of 6-gingerol against DSS-induced colitis. We concluded that 6-gingerol exerts anti-inflammatory effects through AMPK activation. It is suggested that 6-gingerol has a promising role in treatment of IBD.

Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway.

PubMed

Zhao, Yingshuai; Wang, Liuyi; He, Shanshan; Wang, Xiaoyan; Shi, Weili

2017-05-20

Valsartan (VAL), an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO). In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs). Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L) were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L), VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L), and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L) groups. The NO content in the VAL-treated HUVEC line (EA.hy926) was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05) and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial.

PubMed

Amsler, K

1990-07-01

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.

Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

PubMed

Yerra, Veera Ganesh; Kumar, Ashutosh

2017-04-01

Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague-Dawley rats (250-270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

An Effect of Dexamethasone on Adenosine 3′,5′ -Monophosphate Content and Adenosine 3′,5′ -Monophosphate Phosphodiesterase Activity of Cultured Hepatoma Cells

PubMed Central

Manganiello, Vincent; Vaughan, Martha

1972-01-01

The effect of dexamethasone on adenosine 3′,5′-monophosphate (cAMP) phosphodiesterase activity in cultured HTC hepatoma cells was investigated. Homogenates of these cells contain phosphodiesterase activity with two apparent Michaelis constants for cAMP (2-5 μm and 50 μm). At all substrate concentrations tested, phosphodiesterase activity was decreased 25-40% in cells incubated for 36 hr or more with 1 μm dexamethasone. Acid phosphatase activity in the same cells was not decreased. α-Methyl testosterone, 1 μm, was without effect on phosphodiesterase activity. Incubation for 10 min with epinephrine plus theophylline increased the cAMP content of the HTC cells 3- to 6-fold. In cells incubated for 72 hr with dexamethasone, the basal concentration of cAMP was slightly increased and the increment produced by epinephrine plus theophylline was markedly increased. We believe that in many cells the so-called permissive effects of steroid hormones on cAMP mediated processes may be due to an effect of these hormones on cAMP phosphodiesterase activity similar to that observed in HTC cells incubated with dexamethasone. PMID:4341439

Hepatic oxidative stress in ovariectomized transgenic mice expressing the hepatitis C virus polyprotein is augmented through suppression of adenosine monophosphate-activated protein kinase/proliferator-activated receptor gamma co-activator 1 alpha signaling.

PubMed

Tomiyama, Yasuyuki; Nishina, Sohji; Hara, Yuichi; Kawase, Tomoya; Hino, Keisuke

2014-10-01

Oxidative stress plays an important role in hepatocarcinogenesis of hepatitis C virus (HCV)-related chronic liver diseases. Despite the evidence of an increased proportion of females among elderly patients with HCV-related hepatocellular carcinoma (HCC), it remains unknown whether HCV augments hepatic oxidative stress in postmenopausal women. The aim of this study was to determine whether oxidative stress was augmented in ovariectomized (OVX) transgenic mice expressing the HCV polyprotein and to investigate its underlying mechanisms. OVX and sham-operated female transgenic mice expressing the HCV polyprotein and non-transgenic littermates were assessed for the production of reactive oxygen species (ROS), expression of inflammatory cytokines and antioxidant potential in the liver. Compared with OVX non-transgenic mice, OVX transgenic mice showed marked hepatic steatosis and ROS production without increased induction of inflammatory cytokines, but there was no increase in ROS-detoxifying enzymes such as superoxide dismutase 2 and glutathione peroxidase 1. In accordance with these results, OVX transgenic mice showed less activation of peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α), which is required for the induction of ROS-detoxifying enzymes, and no activation of adenosine monophosphate-activated protein kinase-α (AMPKα), which regulates the activity of PGC-1α. Our study demonstrated that hepatic oxidative stress was augmented in OVX transgenic mice expressing the HCV polyprotein by attenuation of antioxidant potential through inhibition of AMPK/PGC-1α signaling. These results may account in part for the mechanisms by which HCV-infected women are at high risk for HCC development when some period has passed after menopause. © 2013 The Japan Society of Hepatology.

[Alteration of metabolic characteristics on the masseter muscle fiber of unilateral chewing rats and its adenosine monophosphate activated protein kinase regulatory mechanism].

PubMed

Andi, Shi; Lin, Zeng; Jing, Liu

2017-06-01

This study aims to determine the influence of unilateral chewing on metabolic characteristics of masseter muscle fibers in rats and the regulatory effect of an adenosine monophosphate activated protein kinase (AMPK) signal pathway on metabolism. Rats were submitted to exodontia of all the right maxillary molars and divided into 2, 4, 6, and 8 weeks groups, and corresponding control groups were set as well. Sections were stained by nicotine adenine dinucleotide tetrazolim reductase(NADH-TRase) to demonstrate the types, proportion, and density of masseter muscle fibers. AMPKα1 and p-AMPK(Thr172) levels in bilateral masseter muscles were detected by Western blot. In the 2-week group, the percentage of dark fibers augmented in the ipsilateral side, whereas the percentage of intermediary fibers in the contralateral side was increased accompanied by a decrease of light fibers, compared with the control group (P<0.05). The percentage of dark fibers was increased in the bilateral sides, whereas the percentage of dark fiber in the ipsilateral sides surpassed that of the contralateral sides in the 4, 6, and 8-week groups. The percentage of intermediary fibers was decreased in the bilateral sides in the 6 and 8-week groups (P<0.05). The percentage of light fibers was reduced in the ipsilateral sides in the 8-week group, whereas no alteration was observed in contralateral sides (P>0.05). In the ipsilateral sides, p-AMPK (Thr172)/AMPKα1 levels were increased in the 2 and 4-week groups (P<0.05), whereas no change was observed in the contralateral sides in either group (P>0.05). Unilateral chewing increases the oxidative metabolic ability in bilateral masseter muscle fibers especially in the non-working side accompanied with change of muscle fiber types. The improvement of aerobic metabolism ability is related to the AMPK signal pathway.
.

The ABCD's of 5'-adenosine monophosphate-activated protein kinase and adrenoleukodystrophy.

PubMed

Weidling, Ian; Swerdlow, Russell H

2016-07-01

This Editorial highlights a study by Singh and coworkers in the current issue of Journal of Neurochemistry, in which the authors present additional evidence that AMPKα1 is reduced in X-linked adrenoleukodystrophy (X-ALD). They make a case for increasing AMPKα1 activity for therapeutic purposes in this disease, and indicate how this goal may be achieved. Read the highlighted article 'Metformin-induced mitochondrial function and ABCD2 up regulation in X-linked adrenoleukodystrophy involves AMP activated protein kinase' on page 86. © 2016 International Society for Neurochemistry.

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway.

PubMed

Tseng, Shih-Ya; Chao, Ting-Hsing; Li, Yi-Heng; Liu, Ping-Yen; Lee, Cheng-Han; Cho, Chung-Lung; Wu, Hua-Lin; Chen, Jyh-Hong

2016-04-01

Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules in vitro and in vivo. Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway in vitro and in vivo. Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

PubMed

First, Eric A

2015-08-15

A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

PubMed

Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

2016-04-01

Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Glycolytic potential and activity of adenosine monophosphate kinase (AMPK), glycogen phosphorylase (GP) and glycogen debranching enzyme (GDE) in steer carcasses with normal (<5.8) or high (>5.9) 24h pH determined in M. longissimus dorsi.

PubMed

Apaoblaza, A; Galaz, A; Strobel, P; Ramírez-Reveco, A; Jeréz-Timaure, N; Gallo, C

2015-03-01

Muscle glycogen concentration (MGC) and lactate (LA), activity of glycogen debranching enzyme (GDE), glycogen phosphorylase (GP) and adenosine monophosphate kinase (AMPK) were determined at 0.5h (T0) and 24h (T24) post-mortem in Longissimus dorsi samples from 38 steers that produced high pH (>5.9) and normal pH (<5.8) carcasses at 24h postmortem. MGC, LA and glycolytic potential were higher (P<0.05) in normal pH carcasses. GDE activity was similar (P>0.05) in both pH categories. GP activity increased between T0 and T24 only in normal pH carcasses. AMPK activity was four times higher in normal pH v/s high pH carcasses, without changing its activity over time. Results reinforce the idea that differences in postmortem glycogenolytic/glycolytic flow in L. dorsi of steers showing normal v/s high muscle pH at 24h, could be explained not only by the higher initial MGC in normal pH carcasses, but also by a high and sustained activity of AMPK and an increased GP activity at 24h postmortem. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

PubMed Central

Ross, Fiona M; Brodie, Martin J; Stone, Trevor W

1998-01-01

When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 μM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5′-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity.ATP can be metabolized by ecto-nucleotidases to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine.We now show that both AMP and ATP (50 μM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5′Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml−1), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 μM), AMP deaminase prevented the depressant effect of AMP on discharge rate.AMP deaminase, at a concentration of 0.2 u ml−1 which annulled the effect of AMP (50 μM), prevented the inhibitory activity of ATP (50 μM). A higher concentration of ATP (200 μM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase.Superfusion of the slices with 5′-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges.The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the

The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

PubMed

Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

2016-10-01

Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) in hypothalamic AMPK and metabolic neuropeptide adaptation to recurring insulin-induced hypoglycemia in the male rat.

PubMed

Mandal, Santosh K; Shrestha, Prem K; Alenazi, Fahaad S H; Shakya, Manita; Alhamami, Hussain; Briski, Karen P

2017-12-01

Glucose counter-regulatory dysfunction correlates with impaired activation of the hypothalamic metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK). Hypothalamic AMPK is controlled by hindbrain energy status; we examined here whether hindbrain AMPK regulates hypothalamic AMPK and metabolic neurotransmitter maladaptation to recurring insulin-induced hypoglycemia (RIIH). Brain tissue was harvested after single versus serial insulin (I) dosing for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant biosynthetic enzyme/neuropeptide expression in micro-punch dissected arcuate (ARH), ventromedial (VMH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissue. The AMPK inhibitor compound c (Cc) or vehicle was administered to the caudal fourth ventricle ahead of antecedent I injections. RIIH caused site-specific elevation (ARH, VMH, LHA) or reduction (DMH) of total AMPK protein versus acute hypoglycemia; Cc respectively exacerbated or attenuated this response in the ARH and VMH. Hindbrain AMPK correspondingly inhibited or stimulated LHA and DMH pAMPK expression during RIIH. RIIH elicited Cc-reversible augmentation of VMH glutamate decarboxylase profiles, but stimulated (ARH pro-opiomelanocortin; LHA orexin-A) or decreased (VMH nitric oxide synthase) other metabolic neurotransmitters without hindbrain sensor involvement. Results demonstrate acclimated up-regulation of total AMPK protein expression in multiple hypothalamic loci during RIIH, and document hindbrain sensor contribution to amplification of this protein profile in the VMH. Concurrent lack of net change in ARH and VMH tissue pAMPK implies adaptive reductions in local sensor activity, which may/may not reflect positive gain in energy state. It remains unclear if 'glucose-excited' VMH GABAergic and/or ARH pro-opiomelanocortin neurons exhibit AMPK habituation to RIIH, and whether diminished sensor activation in these and other mediobasal hypothalamic neurotransmitter

Effects of adenosine 5'-monophosphate on epidermal turnover.

PubMed

Furukawa, Fukumi; Kanehara, Shoko; Harano, Fumiki; Shinohara, Shigeo; Kamimura, Junko; Kawabata, Shigekatsu; Igarashi, Sachiyo; Kawamura, Mitsuaki; Yamamoto, Yuki; Miyachi, Yoshiki

2008-10-01

The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

Crystal structures of the adenylate sensor from fission yeast AMP-activated protein kinase.

PubMed

Townley, Robert; Shapiro, Lawrence

2007-03-23

The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular ATP (adenosine triphosphate) and AMP concentrations. Here, we report crystal structures at 2.9 and 2.6 A resolution for ATP- and AMP-bound forms of a core alphabetagamma adenylate-binding domain from the fission yeast AMPK homolog. ATP and AMP bind competitively to a single site in the gamma subunit, with their respective phosphate groups positioned near function-impairing mutants. Unexpectedly, ATP binds without counterions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells.

PubMed

Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

2014-03-25

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5'-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a 'calm down' signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001.

Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

PubMed Central

Leitch, G J; Amer, M S

1975-01-01

Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed. PMID:164410

Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

PubMed

Liu, Hong; Zhang, Yujin; Wu, Hongyu; D'Alessandro, Angelo; Yegutkin, Gennady G; Song, Anren; Sun, Kaiqi; Li, Jessica; Cheng, Ning-Yuan; Huang, Aji; Edward Wen, Yuan; Weng, Ting Ting; Luo, Fayong; Nemkov, Travis; Sun, Hong; Kellems, Rodney E; Karmouty-Quintana, Harry; Hansen, Kirk C; Zhao, Bihong; Subudhi, Andrew W; Jameson-Van Houten, Sonja; Julian, Colleen G; Lovering, Andrew T; Eltzschig, Holger K; Blackburn, Michael R; Roach, Robert C; Xia, Yang

2016-08-02

High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Together, human and mouse studies reveal novel

Dan-gua fang improves glycolipid metabolic disorders by promoting hepatic adenosine 5'-monophosphate activated protein kinase expression in diabetic Goto-Kakizaki rats.

PubMed

Lan, Yuan-long; Huang, Su-ping; Heng, Xian-pei; Chen, Ling; Li, Peng-hui; Wu, Jing; Yang, Liu-qing; Pan, Xu-dong; Lin, Tong; Cheng, Xin-ling; Lin, Qing; Chen, Si-xin

2015-03-01

To investigate the effect of Dan-gua Fang on adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK) α expression in liver and subsequent improvement of glucose and lipid metabolism. Forty 13-week-old diabetic Goto-Kakizaki (GK) rats were randomly divided into model, Dan-gua Fang, metformin and simvastatin groups (n=10 for each), and fed high-fat diet ad libitum. Ten Wistar rats were used as normal group and fed normal diet. After 24 weeks, liver expression of AMPKα mRNA was assessed by real-time PCR. AMPKα and phospho-AMPKα protein expression in liver was evaluated by Western blot. Liver histomorphology was carried out after hematoxylin-eosin staining, and blood glucose (BG), glycosylated hemoglobin A1c (HbA1c), food intake and body weight recorded. Similar AMPKα mRNA levels were found in the Dan-gua Fang group and normal group, slightly higher than the values obtained for the remaining groups (P<0.05). AMPKα protein expression in the Dan-gua Fang group animals was similar to other diabetic rats, whereas phospho-AMPKα (Thr-172) protein levels were markedly higher than in the metformin group and simvastatin group (P<0.05), respectively. However, phosphor-AMPKα/AMPKα ratios were similar in all groups. Dan-gua Fang reduced fasting blood glucose with similar strength to metformin, and was superior in reducing cholesterol, triglycerides, high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin. Dan-gua Fang decreases plasma alanine aminotransferase (ALT) significantly. Dan-gua Fang, while treating phlegm-stasis, could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet, and effectively protect liver histomorphology and function. This may be partly explained by increased AMPK expression in liver. Therefore, Dan-gua Fang might be an ideal drug for comprehensive intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.

Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

PubMed Central

Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

2014-01-01

Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP.

PubMed

Wu, Jian; Jones, John M; Nguyen-Huu, Xuong; Ten Eyck, Lynn F; Taylor, Susan S

2004-06-01

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced

Early glycogen synthase kinase-3β and protein phosphatase 2A independent tau dephosphorylation during global brain ischaemia and reperfusion following cardiac arrest and the role of the adenosine monophosphate kinase pathway.

PubMed

Majd, Shohreh; Power, John H T; Koblar, Simon A; Grantham, Hugh J M

2016-08-01

Abnormal tau phosphorylation (p-tau) has been shown after hypoxic damage to the brain associated with traumatic brain injury and stroke. As the level of p-tau is controlled by Glycogen Synthase Kinase (GSK)-3β, Protein Phosphatase 2A (PP2A) and Adenosine Monophosphate Kinase (AMPK), different activity levels of these enzymes could be involved in tau phosphorylation following ischaemia. This study assessed the effects of global brain ischaemia/reperfusion on the immediate status of p-tau in a rat model of cardiac arrest (CA) followed by cardiopulmonary resuscitation (CPR). We reported an early dephosphorylation of tau at its AMPK sensitive residues, Ser(396) and Ser(262) after 2 min of ischaemia, which did not recover during the first two hours of reperfusion, while the tau phosphorylation at GSK-3β sensitive but AMPK insensitive residues, Ser(202) /Thr(205) (AT8), as well as the total amount of tau remained unchanged. Our data showed no alteration in the activities of GSK-3β and PP2A during similar episodes of ischaemia of up to 8 min and reperfusion of up to 2 h, and 4 weeks recovery. Dephosphorylation of AMPK followed the same pattern as tau dephosphorylation during ischaemia/reperfusion. Catalase, another AMPK downstream substrate also showed a similar pattern of decline to p-AMPK, in ischaemic/reperfusion groups. This suggests the involvement of AMPK in changing the p-tau levels, indicating that tau dephosphorylation following ischaemia is not dependent on GSK-3β or PP2A activity, but is associated with AMPK dephosphorylation. We propose that a reduction in AMPK activity is a possible early mechanism responsible for tau dephosphorylation. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Adenosine 5'-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice.

PubMed

Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

2014-01-09

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5'-monophosphate (5'-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5'-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5'-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5'-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5'-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury.

Magnesium Lithospermate B Implicates 3'-5'-Cyclic Adenosine Monophosphate/Protein Kinase A Pathway and N-Methyl-d-Aspartate Receptors in an Experimental Traumatic Brain Injury.

PubMed

Chang, Chih-Zen; Wu, Shu-Chuan; Kwan, Aij-Lie; Lin, Chih-Lung

2015-10-01

Decreased 3'-5'-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and increased N-methyl-d-aspartate (NMDA) related apoptosis were observed in traumatic brain injury (TBI). It is of interest to examine the effect of magnesium lithospermate B (MLB) on cAMP/PKA pathway and NMDAR in TBI. A rodent weight-drop TBI model was used. Administration of MLB was initiated 1 week before (precondition) and 24 hours later (reversal). Cortical homogenates were harvested to measure cAMP (enzyme-linked immunosorbent assay), soluble guanylyl cyclases, PKA and NMDA receptor-2β (Western blot). In addition, cAMP kinase antagonist and H-89 dihydrochloride hydrate were used to test MLB's effect on the cytoplasm cAMP/PKA pathway after TBI. Morphologically, vacuolated neuron and activated microglia were observed in the TBI groups but absent in the MLB preconditioning and healthy controls. Induced cAMP, soluble guanylyl cyclase α1, and PKA were observed in the MLB groups, when compared with the TBI group (P < 0.01) Administration of H-89 dihydrochloride hydrate reversed the effect of MLB on cortical PKA and NMDA-2β expression after TBI. This study showed that MLB exerted an antioxidant effect on the enhancement of cytoplasm cAMP and PKA. This compound also decreased NMDA-2β levels, which may correspond to its neuroprotective effects. This finding lends credence to the presumption that MLB modulates the NMDA-2β neurotoxicity through a cAMP-dependent mechanism in the pathogenesis of TBI. Copyright © 2015 Elsevier Inc. All rights reserved.

Purification and properties of adenosine kinase from rat brain.

PubMed

Yamada, Y; Goto, H; Ogasawara, N

1980-12-04

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

Adenosine kinase regulation of cardiomyocyte hypertrophy

PubMed Central

Fassett, John T.; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie

2011-01-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKTSer473 phosphorylation but did attenuate sustained phosphorylation of RafSer338 (24–48 h), mTORSer2448 (24–48 h), p70S6kThr389 (2.5–48 h), and ERKThr202/Tyr204 (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6kThr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERKThr202/Tyr204 and AKTSer473. Reduction of Raf-induced p70S6kThr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of

Adenosine kinase regulation of cardiomyocyte hypertrophy.

PubMed

Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

2011-05-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser⁴⁷³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³⁸) (24-48 h), mTOR(Ser²⁴⁴⁸) (24-48 h), p70S6k(Thr³⁸⁹) (2.5-48 h), and ERK(Thr²⁰²/Tyr²⁰⁴) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser⁴⁷³). Reduction of Raf-induced p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.

PubMed

Qin, Qining; Downey, James M; Cohen, Michael V

2003-02-01

Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.

Effects of protopine on blood platelet aggregation. II. Effect on metabolic system of adenosine 3',5'-cyclic monophosphate in platelets.

PubMed

Shiomoto, H; Matsuda, H; Kubo, M

1990-08-01

The mode of action of protopine on rabbit platelet aggregation was investigated in the metabolic system of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in vitro experimental models. The inhibitory activity of protopine on adenosine 5'-diphosphate induced platelet aggregation was increased in the presence of prostaglandin I2 or papaverine in platelets. Protopine elevated content of the basal cyclic AMP accumulation in platelets and enhanced activity of crude adenylate cyclase prepared from platelets, but was ineffective on cyclic AMP phosphodiesterase. It is concluded that protopine has an inhibitory activity on platelet aggregation, activates adenylate cyclase and increases cyclic AMP content in platelets, in addition to other inhibitory actions in the metabolic system of cyclic AMP.

Ticagrelor Compared with Clopidogrel Increased Adenosine and Cyclic Adenosine Monophosphate Plasma Concentration in Acute Coronary Syndrome Patients.

PubMed

Li, Xiaoye; Wang, Qibing; Xue, Ying; Chen, Jiahui; Lv, Qianzhou

2017-06-01

Ticagrelor produces a more potent antiplatelet effect than clopidogrel and inhibits cellular uptake of adenosine, which is associated with several cardiovascular consequences. We aimed to explore the correlation between adenosine and cyclic adenosine monophosphate (cAMP) plasma concentration and antiplatelet effect by clopidogrel or ticagrelor in patients with acute coronary syndrome (ACS) receiving dual antiplatelet therapy (DAPT). We conducted a prospective, observational and single-centre cohort study enrolling 68 patients with non-ST-segment elevation ACS from January 2016 to May 2016. We monitored the inhibition of platelet aggregation (IPA) and assessed adenosine, adenosine deaminase (ADA) and cAMP plasma concentrations by immunoassay on admission and 48 hr after coronary angiography. The demographic and clinical data were collected by reviewing their medical records. The two groups exhibited similar baseline characteristics including adenosine, ADA and cAMP. The mean IPA in patients receiving ticagrelor was significantly higher than that in patients receiving clopidogrel (93.5% versus 67.2%; p = 0.000). Also, we observed that patients treated with ticagrelor had a significantly higher increase in levels of adenosine and cAMP compared with those treated with clopidogrel (1.04 (0.86; 1.41) versus 0.04 (-0.25; 0.26); p = 0.029 and 0.78 (-1.67; 1.81) versus 0.60 (-1.91; 4.60); p = 0.037, respectively). And there was a weak correlation between IPA and adenosine as well as cAMP plasma concentration (r = 0.390, p = 0.001 and r = 0.335, p = 0.005, respectively). Ticagrelor increased adenosine and cAMP plasma concentration compared with clopidogrel in patients with ACS. © 2017 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

A High Affinity Adenosine Kinase from Anopheles gambiae

PubMed Central

Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

2011-01-01

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

A High-Affinity Adenosine Kinase from Anopheles Gambiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Cassera; M Ho; E Merino

2011-12-31

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactionsmore » for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.« less

3-phosphoglycerate kinase from Hydrogenomonas facilis.

NASA Technical Reports Server (NTRS)

Mcfadden, B. A.; Schuster, E.

1972-01-01

Description of studies of the kinetics of heat inactivation of phosphoglycerate kinase in the soluble fraction from Hydrogenomonas facilis, its extensive purification, and inhibition by adenosine monophosphate (AMP). No evidence was found for an enzyme which catalyzes adenosine-triphosphate-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate.

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

PubMed

Bhaumik, D; Datta, A K

1988-04-01

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

PubMed Central

Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

2014-01-01

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

PubMed Central

Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

2016-01-01

Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

PubMed

Davidson, Jesse A; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan; Wischmeyer, Paul E; Klawitter, Jelena

2016-01-01

Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

PubMed

Smith, N; Broadley, K J

2008-09-01

Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

[Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate].

PubMed

Medentsev, A G; Arinbasarova, A Iu; Smirnova, N M; Akimenko, V K

2004-01-01

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5'-monophosphorothioate.

PubMed Central

Fedan, J. S.

1987-01-01

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter. PMID:3038248

Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

PubMed Central

Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

2012-01-01

SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

Involvement of adenosine monophosphate activated kinase in interleukin-6 regulation of steroidogenic acute regulatory protein and cholesterol side chain cleavage enzyme in the bovine zona fasciculata and zona reticularis.

PubMed

De Silva, Matharage S I; Dayton, Adam W; Rhoten, Lance R; Mallett, John W; Reese, Jared C; Squires, Mathieu D; Dalley, Andrew P; Porter, James P; Judd, Allan M

2018-06-01

In bovine adrenal zona fasciculata (ZF) and NCI-H295R cells, interleukin-6 (IL-6) increases cortisol release, increases expression of steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (P450scc), and steroidogenic factor 1 (SF-1) (increases steroidogenic proteins), and decreases the expression of adrenal hypoplasia congenita-like protein (DAX-1) (inhibits steroidogenic proteins). In contrast, IL-6 decreases bovine adrenal zona reticularis (ZR) androgen release, StAR, P450scc, and SF-1 expression, and increases DAX-1 expression. Adenosine monophosphate (AMP) activated kinase (AMPK) regulates steroidogenesis, but its role in IL-6 regulation of adrenal steroidogenesis is unknown. In the present study, an AMPK activator (AICAR) increased (P < 0.01) NCI-H295R StAR promoter activity, StAR and P450scc expression, and the phosphorylation of AMPK (PAMPK) and acetyl-CoA carboxylase (PACC) (indexes of AMPK activity). In ZR (decreased StAR, P450scc, SF-1, increased DAX-1) (P < 0.01) and ZF tissues (increased StAR, P450scc, SF-1, decreased DAX-1) (P < 0.01), AICAR modified StAR, P450scc, SF-1 and DAX-1 mRNAs/proteins similar to the effects of IL-6. The activity (increased PAMPK and PACC) (P < 0.01) of AMPK in the ZF and ZR was increased by AICAR and IL-6. In support of an AMPK role in IL-6 ZF and ZR effects, the AMPK inhibitor compound C blocked (P < 0.01) the effects of IL-6 on the expression of StAR, P450scc, SF-1, and DAX-1. Therefore, IL-6 modification of the expression of StAR and P450scc in the ZF and ZR may involve activation of AMPK and these changes may be related to changes in the expression of SF-1 and DAX-1. Copyright © 2018 Elsevier Inc. All rights reserved.

Adenosine Monophosphate-Based Detection of Bacterial Spores

NASA Technical Reports Server (NTRS)

Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

2009-01-01

A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

PubMed

Firsov, Alexander M; Rybalkina, Irina G; Kotova, Elena A; Rokitskaya, Tatyana I; Tashlitsky, Vadim N; Korshunova, Galina A; Rybalkin, Sergei D; Antonenko, Yuri N

2018-02-01

The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases, from kinase to sirtuin inhibition.

PubMed

Trapp, Johannes; Jochum, Anne; Meier, Rene; Saunders, Laura; Marshall, Brett; Kunick, Conrad; Verdin, Eric; Goekjian, Peter; Sippl, Wolfgang; Jung, Manfred

2006-12-14

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate.

PubMed

Florentinus-Mefailoski, Angelique; Soosaipillai, Antonius; Dufresne, Jaimie; Diamandis, Eleftherios P; Marshall, John G

2015-02-01

An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was measured to the femtomole range by dilution and direct analysis with micro-liquid chromatography, electrospray ionization, and mass spectrometry (LC-ESI-MS). The LC-ESI-MS assay for adenosine production was shown to be linear and accurate using internal (13)C(15)N adenosine isotope dilution, internal (13)C(15)N adenosine one-point calibration, and external adenosine standard curves with close agreement. The detection limits of LC-ESI-MS for alkaline phosphatase-streptavidin (AP-SA, ∼190,000 Da) was tested by injecting 0.1 μl of a 1 pg/ml solution, i.e., 100 attograms or 526 yoctomole (5.26E-22) of the alkaline-phosphatase labeled probe on column (about 315 AP-SA molecules). The ELIMSA for PSA was linear and showed strong signals across the picogram per milliliter range and could robustly detect PSA from all of the prostatectomy patients and all of the female plasma samples that ranged as low as 70 pg/ml with strong signals well separated from the background and well within the limit of quantification of the AP-SA probe. The results of the ELIMSA assay for PSA are normal and homogenous when independently replicated with a fresh standard over multiple days, and intra and inter diem assay variation was less than 10 % of the mean. In a blind comparison, ELIMSA showed excellent agreement with, but was more sensitive than, the present gold standard commercial fluorescent ELISA, or ECL-based detection, of PSA from normal and prostatectomy samples, respectively.

Synthesis and Release of Cyclic Adenosine 3′:5′-Monophosphate by Ochromonas malhamensis1

PubMed Central

Bressan, Ray A.; Handa, Avtar K.; Quader, Hartmut; Filner, Philip

1980-01-01

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [32P]cAMP was purified from cultures supplied [32P]phosphate. The compound was identified as [32P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a 32P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a 32P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [3H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step. Images PMID:16661154

Effect of bucladesine, pentoxifylline, and H-89 as cyclic adenosine monophosphate analog, phosphodiesterase, and protein kinase A inhibitor on acute pain.

PubMed

Salehi, Forouz; Hosseini-Zare, Mahshid S; Aghajani, Haleh; Seyedi, Seyedeh Yalda; Hosseini-Zare, Maryam S; Sharifzadeh, Mohammad

2017-08-01

The aim of this study was to determine the effects of cyclic adenosine monophosphate (cAMP) and its dependent pathway on thermal nociception in a mouse model of acute pain. Here, we studied the effect of H-89 (protein kinase A inhibitor), bucladesine (Db-cAMP) (membrane-permeable analog of cAMP), and pentoxifylline (PTX; nonspecific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1, and 0.5 mg/100 g), PTX (5, 10, and 20 mg/100 g), and Db-cAMP (50, 100, and 300 nm/mouse) were administered intraperitoneally (I.p.) 15 min before a tail-flick test. In combination groups, we injected the first and the second compounds 30 and 15 min before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Db-cAMP, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100 g) attenuated the antinociceptive effect of Db-cAMP in doses of 50 and 100 nm/mouse. Surprisingly, Db-cAMP decreased the antinociceptive effect of the lowest dose of H-89 (0.05 mg/100 g). All applied doses of PTX reduced the effect of 0.05 mg/100 g H-89 on pain sensation; however, the highest dose of H-89 compromised the antinociceptive effect of 20 mg/100 g dose of PTX. Co-administration of Db-cAMP and PTX increased the antinociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases. © 2017 Société Française de Pharmacologie et de Thérapeutique.

Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside ▿ †

PubMed Central

Chen, Yen-Liang; Yin, Zheng; Duraiswamy, Jeyaraj; Schul, Wouter; Lim, Chin Chin; Liu, Boping; Xu, Hao Ying; Qing, Min; Yip, Andy; Wang, Gang; Chan, Wai Ling; Tan, Hui Pen; Lo, Melissa; Liung, Sarah; Kondreddi, Ravinder Reddy; Rao, Ranga; Gu, Helen; He, Handan; Keller, Thomas H.; Shi, Pei-Yong

2010-01-01

We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis. PMID:20457821

Dielectric spectra broadening as a signature for dipole-matrix interaction. III. Water in adenosine monophosphate/adenosine-5'-triphosphate solutions.

PubMed

Puzenko, Alexander; Levy, Evgeniya; Shendrik, Andrey; Talary, Mark S; Caduff, Andreas; Feldman, Yuri

2012-11-21

In this, the third part of our series on the dielectric spectrum symmetrical broadening of water, we consider the nucleotide aqueous solutions. Where in Parts I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)] and II [E. Levy et al., J. Chem. Phys. 136, 114503 (2012)], the dipole-dipole or ion-dipole interaction had a dominant feature, now the interplay between these two types of dipole-matrix interactions will be considered. We present the results of high frequency dielectric measurements of different concentrations of adenosine monophosphate/adenosine-5'-triphosphate aqueous solutions. We observed the Cole-Cole broadening of the main relaxation peak of the solvent in the solutions. Moreover, depending on the nucleotide concentration, we observed both types of dipole-matrix interaction. The 3D trajectory approach (described in detail in Part I) is applied in order to highlight the differences between the two types of interaction.

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases.

PubMed

Feng, Feng; Yang, Fan; Rong, Wei; Wu, Xiaogang; Zhang, Jie; Chen, She; He, Chaozu; Zhou, Jian-Min

2012-04-15

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.

STUDIES ON THE MECHANISM OF ACTION OF CYCLIC 3’,5’-ADENOSINE MONOPHOSPHATE ON STEROID HYDROXYLATIONS IN ADRENAL HOMOGENATES,

DTIC Science & Technology

Cyclic 3’,5’-adenosine monophosphate (cyclic 3’,5’AMP) has recently been shown to stimulate selectively steroid C-11- beta hydroxylase activity in rat...to be mediated via stimulation of alpha- glucan phosphorylase, which in turn led to enhanced production of G-6-P from glycogen and a concomitant...increase in NADPH generation. However, if cyclic 3’,5’-AMP stimulated steroid 11- beta -hydroxylation in adrenal homogenates only by this mechanism, its

Charge transfer complexes of adenosine-5‧-monophosphate and cytidine-5‧-monophosphate with water-soluble cobalt(II) Schiff base complexes in aqueous solution

NASA Astrophysics Data System (ADS)

Boghaei, Davar M.; Gharagozlou, Mehrnaz

2006-01-01

Water-soluble cobalt(II) tetradentate Schiff base complexes have been shown to form charge transfer (CT) complexes with a series of nucleoside monophosphates including adenosine-5‧-monophosphate (AMP) and cytidine-5‧-monophosphate (CMP). The investigated water-soluble cobalt(II) Schiff base complexes are (i) disodium[{bis(5-sulfo-salicylaldehyde)-o-phenylenediiminato}cobalt(II)], Na2[Co(SO3-salophen)] (1); (ii) disodium[{bis(5-sulfo-salicylaldehyde)-4,5-dimethyl-o-phenylenediiminato}cobalt(II)], Na2[Co(SO3-sal-4,5-dmophen)] (2) and (iii) disodium[{bis(4-methoxy-5-sulfo-salicylaldehyde)-4,5-dimethyl-o-phenylenediiminato}cobalt(II)], Na2[Co(SO3-4-meosal-4,5-dmophen)] (3). The formation constant and thermodynamic parameters for charge transfer complex formation of water-soluble cobalt(II) Schiff base complexes with nucleoside monophosphates were determined spectrophotometrically in aqueous solution at constant ionic strength (I = 0.2 mol dm-3 KNO3) under physiological condition (pH 7.0) and at various temperatures between 288 and 308 K. The stoichiometry has been found to be 1:1 (water-soluble cobalt(II) Schiff base complex: nucleoside monophosphate) in each case. Our spectroscopic and thermodynamic results show that the interaction of water-soluble cobalt(II) Schiff base complexes with the investigated nucleoside monophosphates occurs mainly through the phosphate group. The trend of the interaction according to the cobalt(II) Schiff base complexes due to electronic and steric factors is as follows: Na2[Co(SO3-salophen)] > Na2[Co(SO3-sal-4,5-dmophen)] > Na2[Co(SO3-4-meosal-4,5-dmophen)]. Also the trend of the interaction of a given cobalt(II) Schiff base complex according to the nucleoside monophosphate is as follows: CMP > AMP.

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

PubMed

Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

2016-09-01

The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. © 2016 by The American Society of Hematology.

Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3′:5′-cyclic monophosphate in androgen action

PubMed Central

Singhal, Radhey L.; Parulekar, M. R.; Vijayvargiya, R.; Robison, G. Alan

1971-01-01

1. The ability of exogenously administered cyclic AMP (adenosine 3′:5′-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as α-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2′-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16–24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized–castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a

Adenosine Kinase Deficiency in the Brain Results in Maladaptive Synaptic Plasticity.

PubMed

Sandau, Ursula S; Colino-Oliveira, Mariana; Jones, Abbie; Saleumvong, Bounmy; Coffman, Shayla Q; Liu, Long; Miranda-Lourenço, Catarina; Palminha, Cátia; Batalha, Vânia L; Xu, Yiming; Huo, Yuqing; Diógenes, Maria J; Sebastião, Ana M; Boison, Detlev

2016-11-30

Adenosine kinase (ADK) deficiency in human patients (OMIM:614300) disrupts the methionine cycle and triggers hypermethioninemia, hepatic encephalopathy, cognitive impairment, and seizures. To identify whether this neurological phenotype is intrinsically based on ADK deficiency in the brain or if it is secondary to liver dysfunction, we generated a mouse model with a brain-wide deletion of ADK by introducing a Nestin-Cre transgene into a line of conditional ADK deficient Adk fl/fl mice. These Adk Δbrain mice developed a progressive stress-induced seizure phenotype associated with spontaneous convulsive seizures and profound deficits in hippocampus-dependent learning and memory. Pharmacological, biochemical, and electrophysiological studies suggest enhanced adenosine levels around synapses resulting in an enhanced adenosine A 1 receptor (A 1 R)-dependent protective tone despite lower expression levels of the receptor. Theta-burst-induced LTP was enhanced in the mutants and this was dependent on adenosine A 2A receptor (A 2A R) and tropomyosin-related kinase B signaling, suggesting increased activation of these receptors in synaptic plasticity phenomena. Accordingly, reducing adenosine A 2A receptor activity in Adk Δbrain mice restored normal associative learning and contextual memory and attenuated seizure risk. We conclude that ADK deficiency in the brain triggers neuronal adaptation processes that lead to dysregulated synaptic plasticity, cognitive deficits, and increased seizure risk. Therefore, ADK mutations have an intrinsic effect on brain physiology and may present a genetic risk factor for the development of seizures and learning impairments. Furthermore, our data show that blocking A 2A R activity therapeutically can attenuate neurological symptoms in ADK deficiency. A novel human genetic condition (OMIM #614300) that is based on mutations in the adenosine kinase (Adk) gene has been discovered recently. Affected patients develop hepatic encephalopathy

Kinetic parameters and renal clearances of plasma adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate in man

PubMed Central

Broadus, Arthur E.; Kaminsky, Neil I.; Hardman, Joel G.; Sutherland, Earl W.; Liddle, Grant W.

1970-01-01

Kinetic parameters and the renal clearances of plasma adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) were evaluated in normal subjects using tritium-labeled cyclic nucleotides. Each tracer was administered both by single, rapid intravenous injection and by constant intravenous infusion, and the specific activities of the cyclic nucleotides in plasma and urine were determined. Both cyclic AMP and cyclic GMP were cleared from plasma by glomerular filtration. The kidney was found to add a variable quantity of endogenous cyclic AMP to the tubular urine, amounting to an average of approximately one-third of the total level of cyclic AMP excreted. Plasma was the source of virtually all of the cyclic GMP excreted. Plasma levels of the cyclic nucleotides appeared to be in dynamic steady state. The apparent volumes of distribution of both nucleotides exceeded extracellular fluid volume, averaging 27 and 38% of body weight for cyclic AMP and cyclic GMP, respectively. Plasma production rates ranged from 9 to 17 nmoles/min for cyclic AMP and from 7 to 13 nmoles/min for cyclic GMP. Plasma clearance rates averaged 668 ml/min for cyclic AMP and 855 ml/min for cyclic GMP. Approximately 85% of the elimination of the cyclic nucleotides from the circulation was due to extrarenal clearance. PMID:5480849

Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation

PubMed Central

Yu, Fa-Xing; Zhang, Yifan; Park, Hyun Woo; Jewell, Jenna L.; Chen, Qian; Deng, Yaoting; Pan, Duojia; Taylor, Susan S.; Lai, Zhi-Chun; Guan, Kun-Liang

2013-01-01

The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo–YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions. PMID:23752589

Histone deacetylases 6 increases the cyclic adenosine monophosphate level and promotes renal cyst growth.

PubMed

Wu, Ming; Mei, Changlin

2016-07-01

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal enhanced cell proliferation and fluid secretion, which are triggered by increased levels of cyclic adenosine monophosphate (cAMP). Cebotaru et al. showed that a HDAC6 inhibitor reduced the cAMP level and inhibited cyst formation in Pkd1 knockout mice, which may become a new potential therapeutic agent for ADPKD. This study also raised several intriguing questions that might advance our understanding of the molecular pathogenesis of ADPKD. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase. adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Renosto, F.; Martin, R.L.; Segel, I.H.

1989-06-05

At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the (S)0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound (/sup 35/S)APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivationmore » of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex.« less

Adenosine 5'-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation.

PubMed

Yang, Xiao; Zhan, Yibei; Sun, Qi; Xu, Xi; Kong, Yi; Zhang, Jianfa

2017-01-24

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5'-monophsphate (5'-AMP). We demonstrated that co-administration of APAP and 5'-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5'-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5'-AMP formulation could prevent APAP-induced hepatotoxicity.

In mammalian muscle, SIRT3 is present in mitochondria and not in the nucleus; and SIRT3 is upregulated by chronic muscle contraction in an adenosine monophosphate-activated protein kinase-independent manner.

PubMed

Gurd, Brendon J; Holloway, Graham P; Yoshida, Yuko; Bonen, Arend

2012-05-01

In selected cell lines, it appears (a) that metabolic stressors induce the translocation of SIRT3 from the nucleus to mitochondria and (b) that SIRT3 may contribute to the regulation of mitochondrial biogenesis and/or fatty acid utilization. We have examined in mammalian muscle (1) the association between SIRT3 protein content and muscle oxidative capacity and mitochondrial fatty acid oxidation, (2) the subcellular location of SIRT3, (3) whether exercise induces the translocation of SIRT3 from the nucleus to the mitochondria, and (4) the response of SIRT3 protein to stressors known to induce mitochondrial biogenesis (chronic muscle stimulation and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside administration). SIRT3 protein displayed hierarchical expression based on oxidative potential of muscle tissues (heart > red > white). In contrast to studies in some cell lines, metabolic stress (exercise) did not induce the translocation of SIRT3 from the nucleus to mitochondria, as SIRT3 was only present in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, not in the nucleus. Chronic stimulation increased muscle mitochondrial content and SIRT3 protein in SS (+33%) and IMF (+27%) mitochondria (P < .05). In contrast, chronic 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside administration, while inducing mitochondrial biogenesis, did not alter SS or IMF mitochondrial SIRT3 protein content. These studies have shown that, in muscle, SIRT3 (a) scales with muscle oxidative capacity and with enzymes regulating fatty acid oxidation, (b) in resting muscle is localized to SS and IMF mitochondria and not nuclei, (c) in contracting muscle is not acutely translocated to mitochondria, and (d) is upregulated with chronic stimulation in an adenosine monophosphate-activated protein kinase-independent manner. Copyright © 2012 Elsevier Inc. All rights reserved.

Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC

PubMed Central

Jin, Hui; Hilaire, Cynthia St.; Huang, Yuting; Yang, Dan; Dmitrieva, Natalia I.; Negro, Alejandra; Schwartzbeck, Robin; Liu, Yangtengyu; Yu, Zhen; Walts, Avram; Davaine, Jean-Michel; Lee, Duck-Yeon; Donahue, Danielle; Hsu, Kevin S.; Chen, Jessica; Cheng, Tao; Gahl, William; Chen, Guibin; Boehm, Manfred

2017-01-01

ACDC (arterial calcification due to deficiency of CD73) is an autosomal recessive disease resulting from loss-of-function mutations in NT5E, which encodes CD73, a 5′-ectonucleotidase that converts extracellular adenosine monophosphate to adenosine. ACDC patients display progressive calcification of lower extremity arteries, causing limb ischemia. Tissue-nonspecific alkaline phosphatase (TNAP), which converts pyrophosphate (PPi) to inorganic phosphate (Pi), and extracellular purine metabolism play important roles in other inherited forms of vascular calcification. Compared to cells from healthy subjects, induced pluripotent stem cell–derived mesenchymal stromal cells (iMSCs) from ACDC patients displayed accelerated calcification and increased TNAP activity when cultured under conditions that promote osteogenesis. TNAP activity generated adenosine in iMSCs derived from ACDC patients but not in iMSCs from control subjects, which have CD73. In response to osteogenic stimulation, ACDC patient–derived iMSCs had decreased amounts of the TNAP substrate PPi, an inhibitor of extracellular matrix calcification, and exhibited increased activation of AKT, mechanistic target of rapamycin (mTOR), and the 70-kDa ribosomal protein S6 kinase (p70S6K), a pathway that promotes calcification. In vivo, teratomas derived from ACDC patient cells showed extensive calcification and increased TNAP activity. Treating mice bearing these teratomas with an A2b adenosine receptor agonist, the mTOR inhibitor rapamycin, or the bisphosphonate etidronate reduced calcification. These results show that an increase of TNAP activity in ACDC contributes to ectopic calcification by disrupting the extracellular balance of PPi and Pi and identify potential therapeutic targets for ACDC. PMID:27965423

Change in single cystathionine β-synthase domain-containing protein from a bent to flat conformation upon adenosine monophosphate binding.

PubMed

Jeong, Byung-Cheon; Park, Si Hoon; Yoo, Kyoung Shin; Shin, Jeong Sheop; Song, Hyun Kyu

2013-07-01

Cystathionine β-synthase (CBS) domains are small intracellular modules that can act as binding domains for adenosine derivatives, and they may regulate the activity of associated enzymes or other functional domains. Among these, the single CBS domain-containing proteins, CBSXs, from Arabidopsis thaliana, have recently been identified as redox regulators of the thioredoxin system. Here, the crystal structure of CBSX2 in complex with adenosine monophosphate (AMP) is reported at 2.2Å resolution. The structure of dimeric CBSX2 with bound-AMP is shown to be approximately flat, which is in stark contrast to the bent form of apo-CBSXs. This conformational change in quaternary structure is triggered by a local structural change of the unique α5 helix, and by moving each loop P into an open conformation to accommodate incoming ligands. Furthermore, subtle rearrangement of the dimer interface triggers movement of all subunits, and consequently, the bent structure of the CBSX2 dimer becomes a flat structure. This reshaping of the structure upon complex formation with adenosine-containing ligand provides evidence that ligand-induced conformational reorganization of antiparallel CBS domains is an important regulatory mechanism. Copyright © 2013 Elsevier Inc. All rights reserved.

A short review on structure and role of cyclic-3',5'-adenosine monophosphate-specific phosphodiesterase 4 as a treatment tool.

PubMed

Eskandari, Nahid; Mirmosayyeb, Omid; Bordbari, Gazaleh; Bastan, Reza; Yousefi, Zahra; Andalib, Alireza

2015-01-01

Cyclic nucleotide phosphodiesterases (PDEs) are known as a super-family of enzymes which catalyze the metabolism of the intracellular cyclic nucleotides, cyclic-3',5'-adenosine monophosphate (cAMP), and cyclic-3',5'-guanosine monophosphate that are expressed in a variety of cell types that can exert various functions based on their cells distribution. The PDE4 family has been the focus of vast research efforts over recent years because this family is considered as a prime target for therapeutic intervention in a number of inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and rheumatoid arthritis, and it should be used and researched by pharmacists. This is because the major isoform of PDE that regulates inflammatory cell activity is the cAMP-specific PDE, PDE4. This review discusses the relationship between PDE4 and its inhibitor drugs based on structures, cells distribution, and pharmacological properties of PDE4 which can be informative for all pharmacy specialists.

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

PubMed

Chew, Bee Lynn; Fisk, Ian D; Fray, Rupert; Tucker, Gregory A; Bodi, Zsuzsanna; Ferguson, Alison; Xia, Wei; Seymour, Graham B

2017-01-01

This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

Protein kinase C is involved in cyclic adenosine monophosphate formation due to PGF2 alpha desensitization in bovine iris sphincter.

PubMed

Tachado, S D; Zhang, Y; Abdel-Latif, A A

1993-05-01

To examine the mechanisms underlying the effects of PGF2 alpha receptor desensitization on agonist-induced second messenger formation and contraction in bovine iris sphincter. Short-term PGF2 alpha receptor desensitization of the bovine iris sphincter was carried out by incubating the tissue in Krebs-Ringer bicarbonate buffer containing 25 microM PGF2 alpha for 45 min at 37 degrees C. The effects of PGF2 alpha and other pharmacologic agents on inositol 1,4,5-triphosphate (IP3) production and cyclic adenosine monophosphate (cAMP) formation in desensitized and nondesensitized tissues were monitored by anion-exchange chromatography and radioimmunoassay. In the isolated bovine iris sphincter, protein kinase C (PKC) is involved in the activation of adenylate cyclase and the desensitization of prostaglandin F2 alpha receptor-mediated responses supported by these findings. (A) Exposure of the tissue to phorbol 12,13-dibutyrate, used to activate PKC, enhanced basal cAMP formation in a dose (EC50 = 8.8 x 10(-8) M) and time (t1/2 = 7.5 min) dependent manner. Phorbol 12,13-dibutyrate increased cAMP levels by twofold and it potentiated the isoproterenol-induced cAMP formation. The biologically inactive phorbol ester, 4 alpha-phorbol had no effect. Staurosporine, a potent PKC inhibitor, inhibited phorbol 12,13-dibutyrate-induced cAMP formation in a dose-dependent manner (IC50 of 0.25 microM). The increase in cAMP levels by phorbol 12,13-dibutyrate results from stimulation of adenylate cyclase, rather than from inhibition of cAMP phosphodiesterase, and it is not mediated through Ca2+ mobilization. Pretreatment of the tissue with phorbol 12,13-dibutyrate inhibited IP3 production in response to PGF2 alpha. (B) Desensitization of the sphincter with PGF2 alpha for 45 min increased cAMP formation and attenuated IP3 production and contraction. The effects of PGF2 alpha desensitization were reversed by pretreatment of the tissue with staurosporine. Down-regulation of PKC prevented the

The formation of novel layered compounds by exfoliation and restacking of cadmium phosphorus trisulphide with the biological molecules adenosine monophosphate and cytidine monophosphate included

NASA Astrophysics Data System (ADS)

Westreich, Philippe

2004-12-01

Exfoliated single layer Cd0.8PS3 has been combined with the biological molecules cytidine monophosphate (CMP) and adenosine monophosphate (AMP) to form the novel restacked compound LixCd 0.8PS3(NMP)z(H2O) y, where N stands for cytidine or adenosine. Composition was determined using energy dispersive X-ray spectroscopy, and the structure of these compounds was studied using X-ray diffraction on oriented films. It was found that for the AMP samples, there is little influence of relative humidity (RH) in the range of 0 to 80%, after which there is a rapid expansion of the interlayer space. In the 0 to 80% range, for (AMP)0.5, a host plane spacing near 19.6 A was found. Electron density calculations on the X-ray diffraction pattern suggest a model for the arrangement of guest AMP molecules between the host layers, with an accompanying water molecule. The calculations also suggest that there is a buckling in the host layer of about +/-0.6 A. For the (CMP)0.3 samples, there is more sensitivity to relative humidity in the 0--80% range, with spacings varying from 20 to 24 A. Much of this variation is gradual, but at around 50% RH, there is a discontinous change in the spacing of about 1.8 A, corresponding to less than the size of a water molecule, that appears to arise from a modification of the CMP conformation. Possible reasons far the differences in the behaviour of the two systems are explored.

Alterations in phosphorylated cyclic adenosine monophosphate response element of binding protein activity: a pathway for fetal alcohol syndrome-related neurotoxicity.

PubMed

Roberson, Robin; Cameroni, Irene; Toso, Laura; Abebe, Daniel; Bissel, Stephanie; Spong, Catherine Y

2009-02-01

Fetal alcohol syndrome (FAS) is the leading cause of a spectrum of preventable nongenetic learning and behavioral disorders. In adult (FAS) mice, we measured phosphorylated cyclic adenosine monophosphate response element of binding protein (pCREB) staining in hippocampal subregions to evaluate a possible mechanism underlying FAS learning deficits. Pregnant C57BL6/J mice were treated on gestational day 8 with alcohol or control (saline). After learning assessment, the offspring were perfused for immunohistochemistry and brain sections probed using SER 133 pCREB antibody. Relative staining density was assessed using National Institutes of Health Image software. Statistical analysis included analysis of variance with P < .05 considered significant. In all hippocampal subregions, pCREB staining was greater in the control animals than in the alcohol-treated group (P < or = .0001). In utero alcohol exposure decreased pCREB activity in hippocampal subregions of adult mice. The dentate gyrus had the most robust cumulative decrease in pCREB staining, suggesting FAS adult learning deficits may correlate to enhanced dentate gyrus neurodegeneration.

Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

PubMed

Yamazaki, Yasuhiro; Yasui, Kenta; Hashizume, Takahiro; Suto, Arisa; Mori, Ayaka; Murata, Yuzuki; Yamaguchi, Masahiko; Ikari, Akira; Sugatani, Junko

2015-10-01

The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery. © 2015 by the American Association for the Study of Liver Diseases.

Recognition of adenosine monophosphate and H2PO4- using zinc ensemble of new hexaphenylbenzene derivative: potential bioprobe and multichannel keypad system.

PubMed

Bhalla, Vandana; Vij, Varun; Kumar, Manoj; Sharma, Parduman Raj; Kaur, Tandeep

2012-02-17

Zinc ensemble of hexaphenylbenzene derivative 3 exhibits sensitive response toward adenosine monophosphate (AMP) and H(2)PO(4)(-) ions. Further, the application of derivative 3 as a multichannel molecular keypad could be realized in the presence of inputs of Zn(2+) ions, H(2)PO(4)(-) ions, and AMP.

Hydrogels Based on Ag+ -Modulated Assembly of 5'-Adenosine Monophosphate for Enriching Biomolecules.

PubMed

Hu, Yuanyuan; Xie, Dong; Wu, Yang; Lin, Nangui; Song, Aixin; Hao, Jingcheng

2017-11-07

Supramolecular hydrogels obtained by combining 5'-adenosine monophosphate (AMP) with Ag + were fabricated in this work. Their gelation capability was enhanced by increasing the concentration of Ag + or decreasing the pH. The gels are very sensitive to light, which endows them with potential applications as visible-light photosensitive materials. Coordination between the nucleobase of AMP and Ag + , as well as π-π stacking of nucleobases, are considered to be the main driving forces for self-assembly. The hydrogels successfully achieved the encapsulation and enrichment of biomolecules. Hydrogen bonding between the amino group of guest molecules and silver nanoparticles along the nanofibers drives the enrichment and is considered to be a crucial interaction. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ON THE MECHANISM OF ACTION OF ADRENOCORTICOTROPIC HORMONE: THE BINDING OF CYCLIC-3′,5′-ADENOSINE MONOPHOSPHATE TO AN ADRENAL CORTICAL PROTEIN*

PubMed Central

Gill, Gordon N.; Garren, Leonard D.

1969-01-01

The binding of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) within the adrenal cortical cell was studied. Cyclic AMP binds specifically to a protein which is associated predominantly with the microsomal fraction of the cell. The binding protein was purified approximately 100-fold. PMID:4308274

Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents

PubMed Central

Pacjuk, Olga; Hernández-Huguet, Silvia; Körner, Johanna; Scherer, Katharina; Richling, Elke

2017-01-01

Background: Phosphodiesterases (PDEs) play a major role in the regulation of cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts (Arbutus unedo, Camellia sinensis, Cynara scolymus, Zingiber officinale) with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit (Arbutus unedo) and tea (Camellia sinensis) extracts did not inhibit PDE markedly. Alternatively, artichoke (Cynara scolymus) extract had a significant inhibitory influence on PDE activity (IC50 = 0.9 ± 0.1 mg/mL) as well as its flavone luteolin (IC50 = 41 ± 10 μM) and 3,4-dicaffeoylquinic acid (IC50 > 1.0 mM). Additionally, the ginger (Zingiber officinale) extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC50 = 1.7 ± 0.2 mg/mL and IC50 > 1.7 mM, respectively). Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC50 = 455 ± 19 μg/mL). Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research. PMID:29113064

Adenosine 5′-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation

PubMed Central

Sun, Qi; Xu, Xi; Kong, Yi; Zhang, Jianfa

2017-01-01

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5′-monophsphate (5′-AMP). We demonstrated that co-administration of APAP and 5′-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5′-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5′-AMP formulation could prevent APAP-induced hepatotoxicity. PMID:28031524

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Hotta, Hak; Sada, Kiyonao

2011-11-01

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

[Development of Fluorescence Resonance Energy Transfer Sensor for Determination of Adenosine Monophosphate in Biological Drug].

PubMed

Dong, Ling-yu; Du, Hong-ming; Wang, Peng; Wang, Li-yun; Li, Yi-ke; Zhai, Hong; Feng, Ting; Wang, Xiang-feng; Zhu, Qiao-you; Xie, Meng-xia

2015-11-01

The biological drug of the calf-blood dialysate has various pharmacological effects. It can promote the oxygen and glucose uptake for the hypoxia cells, and has beneficial effects on the malfunction of the blood circulation and trophic disturbances in the brain, and the impairment of peripheral blood circulation. Furthermore, it is favorable to wound healing and can regulate the central nervous system. Adenosine monophosphate (AMP) is a main active ingredient of the biological drug. In this report, a fluorescence resonance energy transfer (FRET) sensor has been developed with β-CD-capped ZnS QDs as energy donor and 3-hydroxyflavone (3-HF) as energy acceptor. The results showed that AMP can lead to the fluorescence quenching of the FRET sensor at 526 nm, and the Stern-Volmer curve between the fluorescence quenching and the concentrations of AMP present a satisfactory linearity with the correlation coefficient of 0.996. The developed sensor has successfully applied for determination of the AMP in the biological drug.

Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

PubMed Central

Chao, Julie; Weathersbee, Carolyn J.

1974-01-01

Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

A defect in KCa3.1 channel activity limits the ability of CD8+ T cells from cancer patients to infiltrate an adenosine-rich microenvironment.

PubMed

Chimote, Ameet A; Balajthy, Andras; Arnold, Michael J; Newton, Hannah S; Hajdu, Peter; Qualtieri, Julianne; Wise-Draper, Trisha; Conforti, Laura

2018-04-24

The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of immunotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibits T cell motility through the A 2A receptor (A 2A R) and suppression of KCa3.1 channels. We conducted three-dimensional chemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8 + T cells from head and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8 + T cells was reduced in the presence of adenosine, and the effect was greater on HNSCC CD8 + T cells than on healthy donor (HD) CD8 + T cells. This response correlated with the inability of CD8 + T cells to infiltrate tumors. The effect of adenosine was mimicked by an A 2A R agonist and prevented by an A 2A R antagonist. We found no differences in A 2A R expression, 3',5'-cyclic adenosine monophosphate abundance, or protein kinase A type 1 activity between HNSCC and HD CD8 + T cells. We instead detected a decrease in KCa3.1 channel activity, but not expression, in HNSCC CD8 + T cells. Activation of KCa3.1 channels by 1-EBIO restored the ability of HNSCC CD8 + T cells to chemotax in the presence of adenosine. Our data highlight the mechanism underlying the increased sensitivity of HNSCC CD8 + T cells to adenosine and the potential therapeutic benefit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Effects of nucleotides adenosine monophosphate and adenosine triphosphate in combination with L-arginine on male rabbit corpus cavernosum tissue.

PubMed

Hupertan, V; Neuzillet, Y; Stücker, O; Pons, C; Leammel, E; Lebret, T

2012-12-01

Purines and more specifically adenosine monophosphate (AMP) and adenosine triphosphate (ATP) have a strong relaxant effect on smooth muscle cells of the dog, rabbit and human corpus cavernosum, to approximately the same degree as nitric oxide (NO). However, purines are considered as modulators of erectile function rather than key mediators. This suggests that the use of purines combined with NO donors could be effective to treat some specific erectile disorders. The relaxation induced by the combination of l-arginine (Arg), a natural substrate for NO synthase, was assessed with a purine-nucleotide (AMP, ATP) on a rabbit corpus cavernosum model, to determine if these substances could potentiate each other's effect. When a pre-contraction was induced by phenylephrine, AMP alone induced a 43% CC relaxation rate and ATP alone a 26% rate. The relaxation rate induced by Arg was lower in comparison (8% at 5.10(-4) m vs. 25% at AMP 5.10(-4) m and 15% at ATP 5.10(-4) m). NO synthase inhibitor n-nitro-l-arginine did not modify the relaxing effect provoked by AMP suggesting that the mechanism of action of this nucleotide does not involve the NO pathway. The combination of Arg at 5.10(-4) m with either AMP or ATP at different doses ranging from 5.10(-4) to 10(-3) m significantly enhanced the relaxing response reaching rates of 62 and 80% respectively, leading to a synergistic effect. The present data indicate that a 'NO donor' combined with an 'adenosine donor' could be an effective therapeutic approach. © 2012 The Authors. International Journal of Andrology © 2012 European Academy of Andrology.

The reproducibility of adenosine monophosphate bronchial challenges in mild, steroid-naive asthmatics

PubMed Central

Singh, Dave; Fairwood, Jennifer; Murdoch, Robert; Weeks, Amanda; Russell, Paul; Roy, Kay; Langley, Steve; Woodcock, Ashley

2008-01-01

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Repeated adenosine monophosphate (AMP) challenges are used to assess drug effects in asthma clinical trials, but may be prone to tachyphylaxis when repeated at short intervals. Possible tachyphylaxis at 12- and 24-h intervals has not been studied. WHAT THIS STUDY ADDS Clinically relevant tachyphylaxis after repeated AMP challenges does not occur when repeated at 12- and 24-h intervals. AMP challenges at these intervals can be used to assess drug effects in clinical trials. AIMS Repeated adenosine monophosphate (AMP) challenges are used to assess drug efficacy in clinical trials of mild, steroid-naive asthmatics. Refractoriness has been reported after repeated challenges over short intervals. This study evaluated possible tachyphylaxis after repeated AMP challenges at 12 and 24 h in mild, steroid-naive asthmatics. METHODS This was an open, three-way crossover study. Twenty-six steroid-naive asthmatic subjects were randomized to the following AMP challenge regimens separated by 7–14 days: (A) challenge at 08.00 h, repeated 24 h later; (B) challenge at 08.00 h, repeated 12 and 24 h later; (C) challenge at 20.00 h, repeated 12 h later. Comparisons within day were assessed using 90% confidence intervals (CIs). Non-inferiority approach taken with 1 doubling concentration (DC) as a clinically relevant difference. RESULTS Regimen A: Significant increase in AMP reactivity at 24 h. Mean DC difference was 0.6 (90% CI 0.24, 0.96). Regimen B: No evidence of difference between AMP reactivity at 08.00 h and a repeated challenge 12 h later. Repeated challenge at 24 h caused a significant increase in provocation concentration (PC)20 compared with 12 h (mean DC difference 0.48, 90% CI 0.02, 0.95) and 0 h (mean DC difference 0.82, 90% CI 0.49, 1.14 – the upper CI exceeds the criteria of 1 DC). Challenge regimen C: No difference between challenges; mean DC difference of 0.28 (90% CI −0.2, 0.76). CONCLUSION The small decline in AMP

Thyroid hormone activates rat liver adenosine 5,-monophosphate-activated protein kinase: relation to CaMKKb, TAK1 and LKB1 expression and energy status.

PubMed

Vargas, R; Ortega, Y; Bozo, V; Andrade, M; Minuzzi, G; Cornejo, P; Fernandez, V; Videla, L A

2013-01-01

AMP-activated protein kinase (AMPK) is a sensor of energy status supporting cellular energy homeostasis that may represent the metabolic basis for 3,3,,5-triiodo-L-thyronine (T3) liver preconditioning. Functionally transient hyperthyroid state induced by T3 (single dose of 0.1 mg/kg) in fed rats led to upregulation of mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of hepatic AMPK at 8 to 36 h after treatment. AMPK Thr 172 phosphorylation induced by T3 is associated with enhanced mRNA expression of the upstream kinases Ca2+ -calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) and transforming growth-factor-beta-activated kinase-1 (TAK1), with increased protein levels of CaMKKbeta and higher TAK1 phosphorylation, without changes in those of the liver kinase B1 (LKB1) signaling pathway. Liver contents of AMP and ADP were augmented by 291 percent and 44 percent by T3 compared to control values (p less than 0.05), respectively, whereas those of ATP decreased by 64% (p less than 0.05), with no significant changes in the total content of adenine nucleotides (AMP + ADP + ATP) at 24 h after T3 administration. Consequently, hepatic ATP/ADP content ratios exhibited 64 percent diminution (p less than 0.05) and those of AMP/ATP increased by 425 percent (p less than 0.05) in T3-treated rats over controls. It is concluded that in vivoT3 administration triggers liver AMPK upregulation in association with significant enhancements in AMPK mRNA expression, AMPK phosphorylation coupled to CaMKKbeta and TAK1 activation, and in AMP/ATP ratios, which may promote enhanced AMPK activity to support T3-induced energy consuming processes such as those of liver preconditioning.

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

PubMed

Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

2015-01-01

Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

PubMed Central

Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

2015-01-01

Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

Effect of nitrogen starvation on the level of adenosine 3',5'-monophosphate in Anabaena variabilis.

PubMed

Hood, E E; Armour, S; Ownby, J D; Handa, A K; Bressan, R A

1979-12-03

Low levels of adenosine 3',5'-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0--2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3--4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development.

Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells.

PubMed

Jung, Seung-Nam; Yang, Woo Kyeom; Kim, Joungmok; Kim, Hak Su; Kim, Eun Ju; Yun, Hee; Park, Hyunsung; Kim, Sung Soo; Choe, Wonchae; Kang, Insug; Ha, Joohun

2008-04-01

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H(2)O(2) and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1alpha protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1alpha ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.

Adenosine monophosphate affects competence development and plasmid DNA transformation in Escherichia coli.

PubMed

Zhang, Yan; Li, Wenhua; Wang, Liming; Shen, Ping; Xie, Zhixiong

2013-11-01

Artificial plasmid DNA transformation of Escherichia coli induced by calcium chloride is a routine technique in molecular biology and genetic engineering processes, but its mechanism has remained elusive. Because adenosine monophosphate (AMP) has been found to regulate natural transformation in Haemophilus influenza, we aimed to investigate the effects of AMP and its derivatives on E. coli transformation by treating competence with different concentrations of them. Analysis of the transformation efficiencies revealed that AMP inhibited the artificial plasmid DNA transformation of E. coli in a concentration- and time-dependent manner. Furthermore, we found that AMP had no effect on the expression of the transformed gene but that the intracellular AMP level of the competent cells rose after a 6 h treatment. These results suggested that the intracellular AMP level had an important role in E. coli transformation. And these have useful implications for the further investigation of the mechanism of E. coli transformation.

Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides.

PubMed

Guo, Li; Breakspear, Andrew; Zhao, Guoyi; Gao, Lixin; Kistler, H Corby; Xu, Jin-Rong; Ma, Li-Jun

2016-02-01

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is a central signalling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signalling in fungal biology has been well documented and the key conserved components, adenylate cyclase (AC) and the catalytic subunit of PKA (CPKA), have been functionally characterized. However, other genes involved in this signalling pathway and their regulation are not well understood in filamentous fungi. Here, we performed a comparative transcriptomics analysis of AC and CPKA mutants in two closely related fungi: Fusarium graminearum (Fg) and F. verticillioides (Fv). Combining available Fg transcriptomics and phenomics data, we reconstructed the Fg cAMP signalling pathway. We developed a computational program that combines sequence conservation and patterns of orthologous gene expression to facilitate global transcriptomics comparisons between different organisms. We observed highly correlated expression patterns for most orthologues (80%) between Fg and Fv. We also identified a subset of 482 (6%) diverged orthologues, whose expression under all conditions was at least 50% higher in one genome than in the other. This enabled us to dissect the conserved and unique portions of the cAMP-PKA pathway. Although the conserved portions controlled essential functions, such as metabolism, the cell cycle, chromatin remodelling and the oxidative stress response, the diverged portions had species-specific roles, such as the production and detoxification of secondary metabolites unique to each species. The evolution of the cAMP-PKA signalling pathway seems to have contributed directly to fungal divergence and niche adaptation. © 2015 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.

Creating Order from Chaos: Cellular Regulation by Kinase Anchoring

PubMed Central

Scott, John D.; Dessauer, Carmen W.; Tasken, Kjetil

2012-01-01

Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems. PMID:23043438

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

PubMed

Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

2017-07-01

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

PubMed

Cabello-Díaz, Juan Miguel; Quiles, Francisco Antonio; Lambert, Rocío; Pineda, Manuel; Piedras, Pedro

2012-04-01

Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

AMP-activated protein kinase at the nexus of therapeutic skeletal muscle plasticity in Duchenne muscular dystrophy.

PubMed

Ljubicic, Vladimir; Jasmin, Bernard J

2013-10-01

Recent studies have highlighted the potential of adenosine monophosphate-activated protein kinase (AMPK) to act as a central therapeutic target in Duchenne muscular dystrophy (DMD). Here, we review the role of AMPK as an important integrator of cell signaling pathways that mediate phenotypic plasticity within the context of dystrophic skeletal muscle. Pharmacological AMPK activation remodels skeletal muscle towards a slower, more oxidative phenotype, which is more pathologically resistant to the lack of dystrophin. Moreover, recent studies suggest that AMPK-activated autophagy may be beneficial for myofiber structure and function in mice with muscular dystrophy. Thus, AMPK may represent an ideal target for intervention because clinically approved pharmacological agonists exist, and because benefits can be derived via two independent yet, complementary biological pathways. The availability of several AMPK activators could therefore lead to the rapid development and implementation of novel and highly effective therapeutics aimed at altering the relentless progression of DMD. Copyright © 2013 Elsevier Ltd. All rights reserved.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

PubMed

Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge

2004-08-01

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

Regulation of cyclic adenosine monophosphate response element binding protein on renin expression in kidney via complex cyclic adenosine monophosphate response element-binding-protein-binding protein/P300 recruitment.

PubMed

Li, Pei; Zhang, Jing; Zhu, Yuanfang; Liu, Ming; Xuan, Jin

2015-11-01

Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene. We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction. With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300. This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.

AMP and adenosine are both ligands for adenosine 2B receptor signaling.

PubMed

Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

2018-01-15

Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

PubMed Central

Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

2016-01-01

Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Browne, E.S.; Bhalla, V.K.

1991-02-01

Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylatedmore » hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.« less

Alpha-Lipoic acid increases energy expenditure by enhancing adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling in the skeletal muscle of aged mice

USDA-ARS?s Scientific Manuscript database

Skeletal muscle mitochondrial dysfunction is associated with aging and diabetes, which decreases respiratory capacity and increases reactive oxygen species. Lipoic acid (LA) possesses antioxidative and antidiabetic properties. Metabolic action of LA is mediated by activation of adenosine monophospha...

Adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

PubMed Central

Lund, Kaleb C.; Wallace, Kendall B.

2008-01-01

Nucleoside analog reverse transcriptase inhibitors (NRTI) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3′-azido-3′-deoxythymidine (AZT; 10 and 50 μM), AZT monophosphate (150 μM), and 2′,3′-dideoxycytidine (ddC; 1μM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2′,3′-dideoxyinosine (ddI; 10 μM) and ddC (1 μM). In the presence of succinate + cAMP AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-γ activity; in the case of AZT these observations may provide a mechanism for the observed long-term toxicity with this drug. PMID:17904600

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

PubMed Central

Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

2013-01-01

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

NASA Technical Reports Server (NTRS)

Mednieks, M. I.; Popova, I.; Grindeland, R. E.

1992-01-01

Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

Adaptive and regulatory mechanisms in aged rats with postoperative cognitive dysfunction

PubMed Central

Bi, Yanlin; Liu, Shuyun; Yu, Xinjuan; Wang, Mingshan; Wang, Yuelan

2014-01-01

Inflammation may play a role in postoperative cognitive dysfunction. 5′ Adenosine monophosphate-activated protein kinase, nuclear factor-kappa B, interleukin-1β, and tumor necrosis factor-α are involved in inflammation. Therefore, these inflammatory mediators may be involved in postoperative cognitive dysfunction. Western immunoblot analysis revealed 5′ adenosine monophosphate-activated protein kinase and nuclear factor-kappa B in the hippocampus of aged rats were increased 1–7 days after splenectomy. Moreover, interleukin-1β and tumor necrosis factor-α were upregulated and gradually decreased. Therefore, these inflammatory mediators may participate in the splenectomy model of postoperative cognitive dysfunction in aged rats. PMID:25206851

Cyclic Adenosine Monophosphate Regulation of Ion Transport in Porcine Vocal Fold Mucosae

PubMed Central

Sivasankar, Mahalakshmi; Nofziger, Charity; Blazer-Yost, Bonnie

2012-01-01

Objectives/Hypothesis Cyclic adenosine monophosphate (cAMP) is an important biological molecule that regulates ion transport and inflammatory responses in epithelial tissue. The present study examined whether the adenylyl cyclase activator, forskolin, would increase cAMP concentration in porcine vocal fold mucosa and whether the effects of increased cAMP would be manifested as a functional increase in transepithelial ion transport. Additionally, changes in cAMP concentrations following exposure to an inflammatory mediator, tumor necrosis factor-α (TNFα) were investigated. Study Design In vitro experimental design with matched treatment and control groups. Methods Porcine vocal fold mucosae (N = 30) and tracheal mucosae (N = 20) were exposed to forskolin, TNFα, or vehicle (dimethyl sulfoxide) treatment. cAMP concentrations were determined with enzyme-linked immunosorbent assay. Ion transport was measured using electrophysiological techniques. Results Thirty minute exposure to forskolin significantly increased cAMP concentration and ion transport in porcine vocal fold and tracheal mucosae. However, 30-minute and 2-hour exposure to TNFα did not significantly alter cAMP concentration. Conclusions We demonstrate that forskolin-sensitive adenylyl cyclase is present in vocal fold mucosa, and further, that the product, cAMP increases vocal fold ion transport. The results presented here contribute to our understanding of the intracellular mechanisms underlying vocal fold ion transport. As ion transport is important for maintaining superficial vocal fold hydration, data demonstrating forskolin-stimulated ion transport in vocal fold mucosa suggest opportunities for developing pharmacological treatments that increase surface hydration. PMID:18596479

Cyclic 3',5'-adenosine monophosphate (cAMP) signaling in the anterior pituitary gland in health and disease.

PubMed

Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

2018-03-05

The cyclic 3',5'-adenosine monophosphate (cAMP) was the first among the so-called "second messengers" to be described. It is conserved in most organisms and functions as a signal transducer by mediating the intracellular effects of multiple hormones and neurotransmitters. In this review, we first delineate how different members of the cAMP pathway ensure its correct compartmentalization and activity, mediate the terminal intracellular effects, and allow the crosstalk with other signaling pathways. We then focus on the pituitary gland, where cAMP exerts a crucial function by controlling the responsiveness of the cells to hypothalamic hormones, neurotransmitters and peripheral factors. We discuss the most relevant physiological functions mediated by cAMP in the different pituitary cell types, and summarize the defects affecting this pathway that have been reported in the literature. We finally discuss how a deregulated cAMP pathway is involved in the pathogenesis of pituitary disorders and how it affects the response to therapy. Copyright © 2017. Published by Elsevier B.V.

Cyclic adenosine 3′,5′-monophosphate in human lymphocytes. Alterations after phytohemagglutinin stimulation

PubMed Central

Smith, Jay W.; Steiner, Alton L.; Newberry, W. Marcus; Parker, Charles W.

1971-01-01

We have studied cyclic adenosine 3′,5′-monophosphate (cyclic AMP) concentrations in human peripheral blood lymphocytes after stimulation with phytohemagglutinin (PHA), isoproterenol, prostaglandins, and aminophylline. Purified lymphocytes were obtained by nylon fiber chromatography, and low speed centrifugation to remove platelets. Cyclic AMP levels were determined by a highly sensitive radioimmunoassay. At concentrations of 0.1-1.0 mmoles/liter isoproterenol and aminophylline produced moderate increases in cyclic AMP concentrations, whereas prostaglandins produced marked elevations. High concentrations of PHA produced 25-300% increases in cyclic AMP levels, alterations being demonstrated within 1-2 min. The early changes in cyclic AMP concentration appear to precede previously reported metabolic changes in PHA-stimulated cells. After 6 hr cyclic AMP levels in PHA-stimulated cells had usually fallen to the levels of control cells. After 24 hr the level in PHA-stimulated cells was characteristically below that of the control cells. Adenyl cyclase, the enzyme which converts ATP to cyclic AMP, was measured in lymphocyte homogenates. Adenyl cyclase activity was rapidly stimulated by fluoride, isoproterenol, prostaglandins, and PHA. Since adenyl cyclase is characteristically localized in external cell membranes, our results are consistent with an initial action of PHA at this level. PMID:4395563

Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

PubMed

Tutton, P J; Barkla, D H

1982-01-01

1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

AMP is an adenosine A1 receptor agonist.

PubMed

Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

2012-02-17

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

PubMed Central

Hatanaka, M; Del Giudice, R; Long, C

1975-01-01

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

PubMed Central

Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

2014-01-01

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2−/−) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2−/− mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2−/−200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism PMID:24968719

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency.

PubMed

Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

2014-08-01

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2(-/-)) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

A step into the RNA world: Conditional analysis of hydrogel formation of adenosine 5'-monophosphate induced by cyanuric acid.

PubMed

Yokosawa, Takumi; Enomoto, Ryota; Uchino, Sho; Hirasawa, Ito; Umehara, Takuya; Tamura, Koji

2017-12-01

Nucleotide polymerization occurs by the nucleophilic attack of 3'-oxygen of the 3'-terminal nucleotide on the α-phosphorus of the incoming nucleotide 5'-triphosphate. The π-stacking of mononucleotides is an important factor for prebiotic RNA polymerization in terms of attaining the proximity of two reacting moieties. Adenosine and adenosine 5'-monophosphate (AMP) are known to form hydrogel in the presence of cyanuric acid at neutral pH. However, we observed that other canonical ribonucleotides did not gel under the same condition. The π-stacking-induced hydrogel formation of AMP was destroyed at pH 2.0, suggesting that the protonation of N at position 1 of adenine abolished hydrogen bonding with the NH of cyanuric acid and resulted in the deformation of the hexad of adenine and cyanuric acid. A liquid-like gel was formed in the case of adenosine with cyanuric acid and boric acid, whereas AMP caused the formation of a solid gel, implying that the negative charge inherent to AMP prevented the formation of esters of boric acid with the cis-diols of ribose. Cyanuric acid-driven oligomerizations of AMP might have been the first crucial event in the foundation of the RNA world. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

PubMed

Schlegel, Nicolas; Baumer, Yvonne; Drenckhahn, Detlev; Waschke, Jens

2009-05-01

To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Experimental laboratory research. Research laboratory. Wistar rats and cultured human microvascular endothelial cells. Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

The adenosine salvage pathway as an alternative to mitochondrial production of ATP in maturing mammalian oocytes.

PubMed

Scantland, Sara; Tessaro, Irene; Macabelli, Carolina H; Macaulay, Angus D; Cagnone, Gaël; Fournier, Éric; Luciano, Alberto M; Robert, Claude

2014-09-01

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation. © 2014 by the Society for the Study of Reproduction, Inc.

AMP Is an Adenosine A1 Receptor Agonist*

PubMed Central

Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

2012-01-01

Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate Effects on Goldfish Behavior and Brain RNA Metabolism

PubMed Central

Shashoua, Victor E.

1971-01-01

Intraventricular administration of dibutyryl adenosine cyclic 3′:5′-monophosphate into goldfish brains produced hyperactive animals. A study of the effects of the drug (25-50 mg/kg) on the incorporation of [5-3H] orotic acid, as a precursor of labeled uridine and cytidine, into newly synthesized RNA showed the formation of an RNA with a uridine to cytidine ratio 20-50% higher than that of the control. In double-labeling experiments with uridine as the labeled precursor, the synthesis of a nuclear RNA fraction (not produced in the absence of drug) was demonstrated. Some of this RNA was found to migrate into the cytoplasmic fraction and to become associated with polysomes. The results suggest that cyclic AMP might function as a “metabolic demand signal” for eliciting new RNA synthesis in goldfish brain. PMID:4330944

Effect of dibutyryl cyclic adenosine monophosphate on the gene expression of plasminogen activator inhibitor-1 and tissue factor in adipocytes.

PubMed

Taniguchi, Makoto; Ono, Naoko; Hayashi, Akira; Yakura, Yuwna; Takeya, Hiroyuki

2011-10-01

Hypertrophic adipocytes in obese states express the elevated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF). An increase in the intracellular concentration of cyclic adenosine monophosphate (cAMP) promotes triglyceride hydrolysis and may improve dysregulation of adipocyte metabolism. Here, we investigate the effect of dibutyryl-cAMP (a phosphodiesterase-resistant analog of cAMP) on the gene expression of PAI-1 and TF in adipocytes. Differentiated 3T3-L1 adipocytes were treated with dibutyryl-cAMP and agents that would be expected to elevate intracellular cAMP, including cilostazol (a phosphodiesterase inhibitor with anti-platelet and vasodilatory properties), isoproterenol (a beta adrenergic agonist) and forskolin (an adenylyl cyclase activator). The levels of PAI-1 and TF mRNAs were measured using real-time quantitative reverse transcription-PCR. The treatment of adipocytes with dibutyryl-cAMP resulted in the inhibition of both lipid accumulation and TF gene expression. However, PAI-1 gene expression was slightly but significantly increased by dibutyryl-cAMP. On the other hand, cilostazol inhibited the expression of PAI-1 without affecting lipid accumulation. When the adipocytes were treated with cilostazol in combination with isoproterenol or forskolin, the inhibitory effect of cilostazol on PAI-1 gene expression was counteracted, thus suggesting that inhibition by cilostazol may not be the result of intracellular cAMP accumulation by phosphodiesterase inhibition. These results suggest the implication of cAMP in regulation of the gene expression of TF and PAI-1 in adipocytes. Our findings will serve as a useful basis for further research in therapy for obesity-associated thrombosis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Regulatory actions of 3',5'-cyclic adenosine monophosphate on osteoclast function: possible roles of Epac-mediated signaling.

PubMed

Jeevaratnam, Kamalan; Salvage, Samantha C; Li, Mengye; Huang, Christopher L-H

2018-05-30

Alterations in cellular levels of the second messenger 3',5'-cyclic adenosine monophosphate ([cAMP] i ) regulate a wide range of physiologically important cellular signaling processes in numerous cell types. Osteoclasts are terminally differentiated, multinucleated cells specialized for bone resorption. Their systemic regulator, calcitonin, triggers morphometrically and pharmacologically distinct retraction (R) and quiescence (Q) effects on cell-spread area and protrusion-retraction motility, respectively, paralleling its inhibition of bone resorption. Q effects were reproduced by cholera toxin-mediated G s -protein activation known to increase [cAMP] i , unaccompanied by the [Ca 2+ ] i changes contrastingly associated with R effects. We explore a hypothesis implicating cAMP signaling involving guanine nucleotide-exchange activation of the small GTPase Ras-proximate-1 (Rap1) by exchange proteins directly activated by cAMP (Epac). Rap1 activates integrin clustering, cell adhesion to bone matrix, associated cytoskeletal modifications and signaling processes, and transmembrane transduction functions. Epac activation enhanced, whereas Epac inhibition or shRNA-mediated knockdown compromised, the appearance of markers for osteoclast differentiation and motility following stimulation by receptor activator of nuclear factor kappa-Β ligand (RANKL). Deficiencies in talin and Rap1 compromised in vivo bone resorption, producing osteopetrotic phenotypes in genetically modified murine models. Translational implications of an Epac-Rap1 signaling hypothesis in relationship to N-bisphosphonate actions on prenylation and membrane localization of small GTPases are discussed. © 2018 New York Academy of Sciences.

Discovery of genetic variants of the kinases that activate tenofovir among individuals in the United States, Thailand, and South Africa: HPTN067.

PubMed

Figueroa, Dominique B; Tillotson, Joseph; Li, Maoji; Piwowar-Manning, Estelle; Hendrix, Craig W; Holtz, Timothy H; Bokoch, Kevin; Bekker, Linda-Gail; van Griensven, Frits; Mannheimer, Sharon; Hughes, James P; Grant, Robert M; Bumpus, Namandjé N

2018-01-01

Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.

Adenosine Kinase Inhibition Protects against Cranial Radiation-Induced Cognitive Dysfunction

PubMed Central

Acharya, Munjal M.; Baulch, Janet E.; Lusardi, Theresa A.; Allen, Barrett. D.; Chmielewski, Nicole N.; Baddour, Al Anoud D.; Limoli, Charles L.; Boison, Detlev

2016-01-01

Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting; however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK). Adult rats exposed to cranial irradiation (10 Gy) showed significant declines in performance of hippocampal-dependent cognitive function tasks [novel place recognition, novel object recognition (NOR), and contextual fear conditioning (FC)] 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the FC task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days) prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS functionality to altered

Abnormal regulation of adenosine 3′,5′-monophosphate and corticosterone formation in an adrenocortical carcinoma

PubMed Central

Ney, R. L.; Hochella, N. J.; Grahame-Smith, D. G.; Dexter, R. N.; Butcher, R. W.

1969-01-01

A spontaneously occurring rat adrenocortical carcinoma which produces corticosterone was maintained by transplantation. The carcinoma appeared to utilize corticosterone biosynthetic steps similar to those of the normal adrenal, but the tumor produced only about 1-10% as much corticosterone per unit tissue weight as nontumorous adrenal glands. The tumor demonstrated little or no increase in corticosterone production in response to adrenocorticotropic hormone (ACTH) either in vivo or in vitro. In normal adrenals, ACTH increases the activity of adenyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to adenosine-3′,5′-monophosphate (cyclic AMP), the latter then serving as an intracellular regulator of steroidogenesis. ACTH failed to increase cyclic AMP levels in the tumor in vivo or in slices in vitro, conditions under which there were 50- and 20-fold increases in nontumorous adrenals. However, in homogenates fortified with exogenous ATP, adenyl cyclase activity was comparable in the tumor and adrenals, and cyclic AMP formation was increased 3-fold by ACTH in each. As measured in homogenates, the tumor did not possess a greater ability to destroy cyclic AMP than did normal adrenals. Although ATP levels in the carcinoma were found to be considerably lower than those in normal adrenals, it was not clear that this finding can explain the inability of ACTH to increase cyclic AMP levels in intact tumor cells. While the failure to normally influence cyclic AMP levels in the carcinoma cells could be an important factor in the lack of a steroid response to ACTH, several lines of evidence suggest that the tumor possesses one or more additional abnormalities in the regulation of steroidogenesis. First, in the absence of ACTH stimulation, the tissue concentrations of cyclic AMP were comparable in the tumor and in nontumorous adrenals, but these cyclic AMP levels were associated with a lower level of steroidogenesis in the tumor. Second, tumor slices

Effects of adenosine monophosphate used in combination with L-arginine on female rabbit corpus cavernosum tissue.

PubMed

Stücker, Olivier; Pons, Catherine; Neuzillet, Yann; Laemmel, Elisabeth; Lebret, Thierry

2014-04-01

Sexual dysfunction is significantly more prevalent in women than in men. However, to date, no satisfactory oral treatment is yet available. The aim of this study was to study the effects of adenosine monophosphate (AMP) alone or its combination with L-Arginine on the relaxation of the female rabbit corpus cavernosum. Cylinder strips from the corporal body of the excised clitoris from female New Zealand White rabbits were incubated in Krebs solution. Phenylephrine (PE) precontraction was achieved, then the drugs AMP and L-Arginine were administered either independently or in sequential combinations to the strips under precontracted conditions. Contraction percentages were compared. When precontraction was induced by PE 8 μM or 20 μM, AMP was shown to induce relaxation up to 25% in a dose-dependent manner. The relaxation induced by L-Arginine reached 15.6% at 5.10(-4) M vs. 16.5% at AMP 5.10(-4) M under the same experimental conditions. Nitric oxide (NO) synthase inhibitor N-nitro-L-arginine strongly inhibited the relaxing effect provoked by AMP, suggesting that the action mechanism of this nucleotide is related to the NO pathway. The combination of L-Arginine at 5.10(-4) M with AMP at different doses ranging from 5.10(-4) M to 10(-3) M significantly amplified the relaxing response up to 40.7% and 58%, respectively. Our results demonstrate that AMP induces a relaxing effect on the female rabbit corpora. They also show that L-Arginine and AMP can potentiate each other and that a synergistic effect can be obtained by their combined use. Because only slight differences exist between both sexes in response to NO donors and/or nucleotide purines or in their use together, it is very likely that close biochemical mechanisms, although not to the same degree and not quite similar, are involved in the engorgement of the penis and the clitoris of New Zealand White rabbits. Stücker O, Pons C, Neuzillet Y, Laemmel E, and Lebret T. Original research-sexual medicine: Effects of

An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep.

PubMed

Bjorness, Theresa E; Dale, Nicholas; Mettlach, Gabriel; Sonneborn, Alex; Sahin, Bogachan; Fienberg, Allen A; Yanagisawa, Masashi; Bibb, James A; Greene, Robert W

2016-03-30

Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to

The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

PubMed Central

Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

2015-01-01

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite.

PubMed

Golitsina, Nina L; Danehy, Francis T; Fellows, Ross; Cretton-Scott, Erika; Standring, David N

2010-03-01

Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2'-C-methyl-cytidine (2'-MeC), entered clinical development as NM283, a valine ester prodrug form of 2'-MeC possessing improved oral bioavailability. To be active against HCV, 2'-MeC must be converted to 2'-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase. Conversion of 2'-MeC to 2'-MeC monophosphate is the first step in 2'-MeC triphosphate production and is thought to be the rate-limiting step. Here we investigate which of three possible enzymes, deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK1), or uridine-cytidine kinase 2 (UCK2), mediate this first phosphorylation step. Purified recombinant enzymes UCK2 and dCK, but not UCK1, could phosphorylate 2'-MeC in vitro. However, siRNA knockdown experiments in three human cell lines (HeLa, Huh7 and HepG2) defined UCK2 and not dCK as the key kinase for the formation of 2'-MeC monophosphate in cultured human cells. These results underscore the importance of confirming enzymatic kinase data with appropriate cell-based assays. Finally, we present data suggesting that inefficient phosphorylation by UCK2 likely limits the antiviral activity of 2'-MeC against HCV. This paves the way for the use of a nucleotide prodrug approach to overcome this limitation.

Cultured astrocytes do not release adenosine during hypoxic conditions

PubMed Central

Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

NASA Astrophysics Data System (ADS)

Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

2015-10-01

This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5'-monophosphate.

PubMed

Shi, Fan; Gong, Shixing; Xu, Li; Zhu, Huanhuan; Sun, Zhenfan; Sun, Wei

2013-12-01

In this paper, a graphene (GR) ionic liquid (IL) 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite-modified carbon molecular wire electrode (CMWE) was fabricated by a drop-casting method and further applied to the sensitive electrochemical detection of adenosine-5'-monophosphate (AMP). CMWE was prepared with diphenylacetylene (DPA) as the modifier and the binder. The properties of modified electrode were examined by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical behaviors of AMP was carefully investigated with enhanced responses appeared, which was due to the presence of GR-IL composite on the electrode surface with excellent electrocatalytic ability. A well-defined oxidation peak of AMP appeared at 1.314 V and the electrochemical parameters were calculated by electrochemical methods. Under the selected conditions, the oxidation peak current of AMP was proportional to its concentration in the range from 0.01 μM to 80.0 μM with the detection limit as 3.42 nM (3σ) by differential pulse voltammetry. The proposed method exhibited good selectivity and was applied to the detection of vidarabine monophosphate injection samples with satisfactory results. © 2013.

Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

PubMed

Kutryb-Zajac, Barbara; Mateuszuk, Lukasz; Zukowska, Paulina; Jasztal, Agnieszka; Zabielska, Magdalena A; Toczek, Marta; Jablonska, Patrycja; Zakrzewska, Agnieszka; Sitek, Barbara; Rogowski, Jan; Lango, Romuald; Slominska, Ewa M; Chlopicki, Stefan; Smolenski, Ryszard T

2016-11-01

Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular

Requirement of the cyclic adenosine monophosphate response element-binding protein for hepatitis B virus replication.

PubMed

Kim, Bo Kyung; Lim, Seoung Ok; Park, Yun Gyu

2008-08-01

The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence-comparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

An adenosine kinase inhibitor attenuates tactile allodynia in a rat model of diabetic neuropathic pain.

PubMed

Lynch, J J; Jarvis, M F; Kowaluk, E A

1999-01-08

The present study was conducted to characterize the development of tactile allodynia in the streptozotocin-induced rat model of diabetes, and to evaluate the antinociceptive effects of systemically administered morphine and the adenosine kinase inhibitor, 5'-deoxy-5-iodotubercidin (5'd-5IT) in this model. Rats were injected with 75 mg/kg streptozotocin (i.p.), and blood glucose levels were determined 3-4 weeks later. Diabetic (blood glucose levels > or = 250 mg/dl) and vehicle-injected rats were examined weekly for the development of tactile allodynia by measuring the threshold for hind paw withdrawal using von Frey hairs. Withdrawal thresholds were reduced to 6.8+/-0.6 g (mean+/-S.E.M.) in approximately one-third of streptozotocin-treated rats 7 weeks after streptozotocin treatment as compared to control thresholds (13.2+/-0.1 g), and this allodynia persisted for at least an additional 7 weeks. In additional experiments, morphine sulfate (5-21 micromol/kg, i.p.) produced dose-dependent antinociceptive effects on tactile allodynia for up to 2 h post-dosing. The adenosine kinase inhibitor, 5'd-5IT (2.5 and 5 micromol/kg, i.p.) also dose-dependently attenuated tactile allodynia. Pretreatment with the opioid receptor antagonist, naloxone (27 micromol/kg, i.p.) or the non-selective adenosine receptor antagonist, theophylline (111 micromol/kg, i.p.) significantly diminished the anti-allodynic effects of morphine and 5'd-5IT, respectively. The present study demonstrates that the potent and selective adenosine kinase inhibitor, 5'd-5IT, is equally effective as morphine in blocking tactile allodynia in this model.

Interactions of 1,12-diamino-4,9-dioxadodecane (OSpm) and Cu(II) ions with pyrimidine and purine nucleotides: adenosine-5'-monophosphate (AMP) and cytidine-5'-monophosphate (CMP).

PubMed

Lomozik, L; Gasowska, A; Krzysko, G

2006-11-01

The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A potentiometric method was applied to determine the composition and stability constants of complexes formed, while the mode of interactions was analysed by spectral methods (ultraviolet and visible spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), (13)C NMR, (31)P NMR). In metal-free systems, molecular complexes nucleotide-polyamine (NMP)H(x)(OSpm) were formed. The endocyclic nitrogen atoms of the purine ring N(1), N(7), the nitrogen atom of the pyrimidine ring N(3), the oxygen atoms of the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine were the reaction centres. The mode of interaction of the metal ion with OSpm and the nucleotides (AMP or CMP) in the coordination compounds was established. In the system Cu(II)/OSpm the dinuclear complex Cu(2)(OSpm) forms, while in the ternary systems Cu(II)/nucleotide/OSpm the species type MH(x)LL' and MLL' appear. In the MH(x)LL' type species, the main centres of copper (II) ion binding in the nucleotide are the phosphate groups. The protonated amino groups of OSpm are involved in non-covalent interaction with the nitrogen atoms N(1), N(7) or N(3) of the purine or pyrimidine ring, whereas at higher pH, deprotonated nitrogen atoms of polyamine are engaged in metallation in MLL' species.

Inhibition of vascular smooth muscle growth via signaling crosstalk between AMP-activated protein kinase and cAMP-dependent protein kinase

PubMed Central

Stone, Joshua D.; Narine, Avinash; Tulis, David A.

2012-01-01

Abnormal vascular smooth muscle (VSM) growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP)-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA). Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remain unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells (VSMC), the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSMC migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashion. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth. PMID:23112775

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

PubMed

Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

2017-08-15

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

PubMed Central

2017-01-01

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding. PMID:28656748

Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.

PubMed

Penpassakarn, Praweenuch; Chaiyen, Pimchai; Palittapongarnpim, Prasit

2011-11-01

Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.

Adenosine Monophosphate Forms Ordered Arrays in Multilamellar Lipid Matrices: Insights into Assembly of Nucleic Acid for Primitive Life

PubMed Central

Toppozini, Laura; Dies, Hannah; Deamer, David W.; Rheinstädter, Maikel C.

2013-01-01

A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5′-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers. PMID:23667523

Adenosine monophosphate forms ordered arrays in multilamellar lipid matrices: insights into assembly of nucleic acid for primitive life.

PubMed

Toppozini, Laura; Dies, Hannah; Deamer, David W; Rheinstädter, Maikel C

2013-01-01

A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5'-adenosine monophosphate (AMP) molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers.

Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases.

PubMed

Vonrhein, C; Schlauderer, G J; Schulz, G E

1995-05-15

There are 17 crystal structures of nucleoside monophosphate kinases known. As expected for kinases, they show large conformational changes upon binding of substrates. These are concentrated in two chain segments, or domains, of 30 and 38 residues that are involved in binding of the substrates N1TP and N2MP (nucleoside tri- and monophosphates with bases N1 and N2), respectively. After aligning the 17 structures on the main parts of their polypeptide chains, two domains in various conformational states were revealed. These states were caused by bound substrate (or analogues) and by crystal-packing forces, and ranged between a 'closed' conformation and a less well defined 'open' conformation. The structures were visually sorted yielding an approximately evenly spaced series of domain states that outlines the closing motions when the substrates bind. The packing forces in the crystals are weak, leaving the natural domain trajectories essentially intact. Packing is necessary, however, to produce stable intermediates. The ordered experimental structures were then recorded as still pictures of a movie and animated to represent the motions of the molecule during a catalytic cycle. The motions were smoothed out by adding interpolated structures to the observed ones. The resulting movies are available through the World Wide Web (http:@bio5.chemie.uni-freiburg.de/ak movie.html). Given the proliferating number of homologous proteins known to exist in different conformational states, it is becoming possible to outline the motions of chain segments and combine them into a movie, which can then represent protein action much more effectively than static pictures alone are able to do.

[The effects of epinephrine and adrenergic antagonists on adenosine 3', 5'-monophosphate level of bovine trabecular cells in vitro].

PubMed

Lu, Y; Li, M; Shen, Y

1998-03-01

To determine the effects of epinephrine (EPI) and adrenergic antagonists on adenosine 3', 5'-monophosphate (cAMP) level of bovine trabecular cells (BTC) in vitro. (3)H-cAMP was used in protein binding assay for measuring the intracellular level of cAMP. (1) 10(-5) mol/L EPI induced a fold increase of cAMP in cultured BTC in vitro; (2) Timilol and ICI 118, 551 blocked efficiently the effect of EPI at a lower concentration (10(-6) mol/L). (3) Bisoprolol did not efficiently block the effect of EPI unless at high concentrations (>or= 10(-5) mol/L). The effects of EPI increasing outflow facility may be associated with its increase of cAMP in trabecular cells; BTC contains beta-adrenergic receptors, and beta(2)-adrenergic receptors are dominant.

AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

PubMed

Neuman, Rugina I; van Kalmthout, Juliette A M; Pfau, Daniel J; Menendez, Dhariyat M; Young, Lawrence H; Forrest, John N

2018-04-01

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr 172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr 172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr 172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

PubMed Central

Hoffmeyer, J.; Neuhard, J.

1971-01-01

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate. PMID:4928005

Adenosine production by human B cells and B cell–mediated suppression of activated T cells

PubMed Central

Saze, Zenichiro; Schuler, Patrick J.; Hong, Chang-Sook; Cheng, Dongmei; Jackson, Edwin K.

2013-01-01

Antibody-independent role of B cells in modulating T-cell responses is incompletely understood. Freshly isolated or cultured B cells isolated from the peripheral blood of 30 normal donors were evaluated for CD39 and CD73 coexpression, the ability to produce adenosine 5′-monophosphate (AMP) and adenosine (ADO) in the presence of exogenous adenosine triphosphate (ATP) as well as A1, A2A, A2B, and A3 adenosine receptor (ADOR) expression. Human circulating B cells coexpress ectonucleotidases CD39 and CD73, hydrolyze exogenous ATP to 5′-AMP and ADO, and express messenger RNA for A1R, A2AR, and A3R. 2-chloroadenosine inhibited B-cell proliferation and cytokine expression, and only A3R selective antagonist restored B-cell functions. This suggested that B cells use the A3R for autocrine signaling and self-regulation. Mediated effects on B-cell growth ± ADOR antagonists or agonists were tested in carboxyfluorescein diacetate succinimidyl ester assays. In cocultures, resting B cells upregulated functions of CD4+ and CD8+ T cells. However, in vitro–activated B cells downregulated CD73 expression, mainly produced 5′-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell–B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 5′-AMP, and ADO. PMID:23678003

Simultaneous determination of 5'-monophosphate nucleotides in infant formulas by HPLC-MS.

PubMed

Ren, Yiping; Zhang, Jingshun; Song, Xiaodan; Chen, Xiaochun; Li, Duo

2011-04-01

A method was developed for simultaneous determination of 5'-monophosphate nucleotides, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, inosine 5'-monophosphate, and uridine 5'-monophosphate in infant formulas by high-performance liquid chromatography-mass spectrometry equipped with electrospray ionization source. The complete chromatographic separation of five nucleotides was achieved through a Symmetry C(18) column, after a binary gradient elution with water containing 0.1% formic acid and acetonitrile as mobile phase. The multi-reaction monitoring mode was applied for tandem mass spectrometry analysis. The established method was further validated by determining the linearity (R(2) > 0.999), recovery (92.0-105.0%), and precision (relative standard deviation ≤6.97%). To verify the applicability of the method, thirty commercially available infant formulas were randomly purchased from the supermarkets in Hangzhou, China, and then analyzed. The results showed that the developed method is validated, sensitive, and reliable for quantitation of nucleotides in infant formulas.

[Role of cyclic adenosine monophosphate response element binding protein in ventricular pacing induced cardiac electrical remodeling in a canine model].

PubMed

Chen, Xuesi; Chen, Xingxing; Cheng, Junhua; Hong, Jun; Zheng, Cheng; Zhao, Jinglin; Li, Jin; Lin, Jiafeng

2015-04-01

This project is designed to explore the potential role of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) in cardiac electrical remodeling induced by pacing at different ventricular positions in dogs. An animal model by implanting the pacemakers in beagles was established. According to the different pacing positions, the animals were divided into 4 groups:conditional control group (n=6), left ventricle pacing group (n=6), right ventricle pacing group (n=6) and bi-ventricle pacing group (n=6). Cardiac and electrical remodeling were observed by echocardiography, electrocardiogram and plasma BNP. Myocardial pathology and protein expression of extracellular regulated protein kinases1/2 (ERK1/2), P38 mitogen activated protein kinases (P38 MAPK) and CREB were examined at 4 weeks post pacing. Cardiac structure and plasma BNP level were similar among 4 groups (all P>0.05). Electrocardiogram derived Tp-Te interval was significantly prolonged post pacing (92±11, 91±10, and 79±13 ms vs. 60±12 ms), and the Tp-Te interval in bi-ventricle pacing group was shorter than in left or right ventricle pacing group (P < 0.05). Western blot results showed that the expression of p-ERK1/2 in left ventricular myocardium of left ventricle pacing group, right ventricular myocardium of right ventricle pacing group and bi-ventricular myocardium of bi-ventricle pacing group was 2.7±0.4, 2.4±0.2, 1.7±0.1 and 1.9±0.2, respectively, the expression of p-P38 MAPK was 1.9±0.3, 1.7±0.2, 0.8±0.1 and 1.1±0.1, respectively, and the expression of p-CREB was 2.1±0.2, 2.0±0.2, 2.7±0.4 and 2.6±0.3, respectively. The p-ERK1/2 and p-P38 MAPK expression of bi-ventricle pacing group was lower,but the p-CREB expression was higher compared to the other pacing groups (P < 0.05). Ventricular pacing could induce electrical remodeling evidenced by prolonged Tp-Te interval and increased phosphorylation of ERK1/2 and p38 MAPK and reduced phosphorylation of CREB. Compared with

Transgenic overexpression of adenosine kinase in brain leads to multiple learning impairments and altered sensitivity to psychomimetic drugs.

PubMed

Yee, Benjamin K; Singer, Philipp; Chen, Jiang-Fan; Feldon, Joram; Boison, Detlev

2007-12-01

The neuromodulator adenosine fulfills a unique role in the brain affecting glutamatergic neurotransmission and dopaminergic signaling via activation of adenosine A1 and A2A receptors, respectively. The adenosine system is thus ideally positioned to integrate glutamatergic and dopaminergic neurotransmission, which in turn could affect behavior and cognition. In the adult brain, adenosine levels are largely regulated by its key metabolic enzyme adenosine kinase (ADK), which may assume the role of an 'upstream regulator' of these two neurotransmitter pathways. To test this hypothesis, transgenic mice with an overexpression of ADK in brain (Adk-tg), and therefore reduced brain adenosine levels, were evaluated in a panel of behavioral and psychopharmacological assays to assess possible glutamatergic and dopaminergic dysfunction. In comparison to non-transgenic control mice, Adk-tg mice are characterized by severe learning deficits in the Morris water maze task and in Pavlovian conditioning. The Adk-tg mice also exhibited reduced locomotor reaction to systemic amphetamine, whereas their reaction to the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 was enhanced. Our results confirmed that ADK overexpression could lead to functional concomitant alterations in dopaminergic and glutamatergic functions, which is in keeping with the hypothesized role of ADK in the balance and integration between glutamatergic and dopaminergic neurotransmission. The present findings are of relevance to current pathophysiological hypotheses of schizophrenia and its pharmacotherapy.

Sensitivity of fructose-1,6-biphosphatase from yeast, liver and skeletal muscle to fructose-2,6-biphosphate and 5'-adenosine monophosphate.

PubMed

von Herrath, M; Holzer, H

1988-05-01

As a prerequisite for future studies on the possible effect of sulphite, an anti-microbial agent, on gluconeogenesis in yeast, a comparative study of fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis, from yeast, liver and skeletal muscle is reported. In contrast to FBPase from yeast or liver, FBPase from skeletal muscle is approximately 1000-fold more sensitive to inhibition by 5' adenosine monophosphate and 30 to 250-fold less sensitive to inhibition by fructose-2,6-bisphosphate. The kinetic properties of the FBPases, determined by the ratios R(Mg2+/Mn2+) and R (pH 7/9) of the enzyme activities, measured at 10 mM Mg2+ and 2 mM Mn2+ and at pH 7.0 and 9.0, respectively, show a drastic difference between the skeletal muscle and the yeast or liver enzymes. The data support the idea that the enzymes from yeast and liver function in gluconeogenesis, whereas the enzyme from skeletal muscle is involved in other biological functions.

Human Protein Kinases and Obesity.

PubMed

Engin, Atilla

2017-01-01

adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. Increase in intracellular oxidative stress can promote PKC-beta activation. Activated PKC-beta induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhances triglyceride accumulation. Obesity is fundamentally caused by cellular energy imbalance and dysregulation. Like adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), N-terminal Per-ARNT-Sim (PAS) kinase are nutrient responsive protein kinases and important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

5'- Adenosine monophosphate induced hypothermia reduces early stage myocardial ischemia/reperfusion injury in a mouse model.

PubMed

Tao, Zhenyin; Zhao, Zhaoyang; Lee, Cheng Chi

2011-08-15

Early intervention using hypothermia treatment has been shown to reduce early inflammation, apoptosis and infarct size in animal models of cardiac ischemia/reperfusion. We have shown that 5'-adenosine monophosphate (5'-AMP) can induce a reversible deep hypothermia in mammals. We hypothesize that 5'-AMP-induced hypothermia (AIH) may reduce ischemic/reperfusion damage following myocardial infarct. C57BL/6J male mice were subjected to myocardial ischemia by ligating the left anterior descending coronary artery (LAD) followed by reperfusion. Compared to euthermic controls, mice given AIH treatment exhibited significant inhibition of neutrophil infiltration and a reduction in matrix metallopeptidase 9 (MMP-9) expressions in the infarcted myocardium. A decrease in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in the left ventricle myocardium were also observed. The overall infarct size of the heart was significantly smaller in AIH treated mice. Myocardial ischemia in mice given 5'-AMP without hypothermia had similar ischemia/reperfusion injuries as the euthermic control. Thus, the AIH cardio-protective effects were primarily hypothermia based.

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

PubMed

Nayar, Utthara; Sadek, Jouliana; Reichel, Jonathan; Hernandez-Hopkins, Denise; Akar, Gunkut; Barelli, Peter J; Sahai, Michelle A; Zhou, Hufeng; Totonchy, Jennifer; Jayabalan, David; Niesvizky, Ruben; Guasparri, Ilaria; Hassane, Duane; Liu, Yifang; Sei, Shizuko; Shoemaker, Robert H; Warren, J David; Elemento, Olivier; Kaye, Kenneth M; Cesarman, Ethel

2017-06-01

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

PubMed

Eltzschig, Holger K; Thompson, Linda F; Karhausen, Jorn; Cotta, Richard J; Ibla, Juan C; Robson, Simon C; Colgan, Sean P

2004-12-15

Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

Total biosynthesis of deoxynucleoside triphosphates using deoxynucleoside monophosphate kinases for PCR application.

PubMed

Bao, Jie; Ryu, Dewey D Y

2007-09-01

Polymerase chain reaction (PCR) and other PCR applications for DNA synthesis require deoxynucleoside triphosphates (dNTP) as the essential precursors and substrates. Currently, the dNTP is commercially produced by a chemical method which is environmentally hazardous and costly due to its low yields in both the synthetic reaction and purification processes. In this study, a enzyme technology for the total integrated biosynthesis of all dNTP components is presented. The bioprocess technology developed and reported here involves two sequential enzymatic phosphorylation reactions coupled with the cofactor regeneration starting from deoxynucleoside monophosphates (dNMP) to deoxynucleoside diphosphates (dNDP) in the first reaction step and to dNTP in the second reaction step in the same bioreactor. The four genes encoding these deoxynucleoside monophosphate kinases were cloned into the recombinant E. coli and expressed using the recombinant E. coli strains. The reaction mechanisms and kinetics of the four kinase enzymes are studied and reported. The total enzymatic syntheses of the four dNTP products were carried out in four separate operations under the high substrate concentrations which emulate the practical application. The optimal process conditions were carefully investigated and complete conversion of dNMP to dNTP at high substrate concentration have been achieved. The purity and quality of dNTP products obtained from this work were analyzed and found to be at least equivalent or better than the commercially available dNTP products. The PCR application of dNTP products obtained from this work were also evaluated for isolating and amplifying genes of different sizes from different organisms. The PCR performance test also showed an equivalent quality as compared to the commercially available dNTP. The bioprocess technology developed and reported here for production of dNTP will provide economically competitive and environmentally friendly viable technology for the

Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

PubMed Central

Wu, Jyun-Yi; Chen, Chia-Hsin; Yeh, Li-Yin; Yeh, Ming-Long; Ting, Chun-Chan; Wang, Yan-Hsiung

2013-01-01

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm−2. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm−2 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm−2 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration. PMID:23788285

Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

PubMed

Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

2015-06-01

Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

A randomized, double-blind, crossover, placebo-controlled comparative clinical trial of arginine aspartate plus adenosine monophosphate for the intermittent treatment of male erectile dysfunction.

PubMed

Neuzillet, Y; Hupertan, V; Cour, F; Botto, H; Lebret, T

2013-03-01

Efficacy and safety of l-arginine aspartate 8 g combined with 200 mg of adenosine monophosphate (AA) with placebo (PL) alone for intermittent treatment of mild-to-moderate erectile dysfunction (ED) were compared. The study design was a double-blind, PL-controlled, two-way crossover randomized clinical trial with 26 patients. Efficacy was assessed by International Index of Erectile Function (IIEF) and two additional validated questionnaires [the Erection Hardness Score (EHS) and the Erectile Dysfunction Inventory of Treatment Satisfaction (EDITS). During each crossover period, separated by a 2-week wash-out period, drugs were administered orally, 1-2 h before sexual intercourse. Primary endpoint was a change in the IIEF. Secondary endpoints were patient and investigator assessments of treatment success. Investigators' and patients' assessment of efficacy was significantly improved by the combination vs. PL (p = 0.01 and p = 0.04 respectively]. EHS and EDITS questionnaires were both improved by the combination (p = 0.015 and p = 0.017 respectively). There was no significant difference in terms of tolerance between AA and PL or severe adverse events. ED patients demonstrated significant improvements in all IIEF domains with the exception of the Sexual Desire and Orgasmic Domains when treated with AA compared with PL. This pilot phase II study showed that the on-demand oral administration at a high dosage of l-arginine aspartate-adenosine monophosphate combination may be effective in patients with mild-to-moderate ED, is very well tolerated and could be tested as a safe first-line therapy in a larger size phase III study. © 2012 American Society of Andrology and European Academy of Andrology.

Hypothermia induced by adenosine 5'-monophosphate attenuates injury in an L-arginine-induced acute pancreatitis rat model.

PubMed

Wang, Yunlong; Guo, Weiting; Li, Yuan; Pan, Xinting; Lv, Wenshan; Cui, Lingling; Li, Changgui; Wang, Yangang; Yan, Shengli; Zhang, Jidong; Liu, Bin

2014-04-01

This study sought to investigate the effects of hypothermia induced by adenosine 5'-monophosphate (5'-AMP) on L-arginine (L-Arg)-induced acute pancreatitis in rats. The rats were divided into four groups: the control group, the acute pancreatitis group, the 5'-AMP pretreatment group, and the 5'-AMP posttreatment group. Rats in all groups, except for the control group, received two injections of 2.5 g/kg body weight (intraperitoneally) L-Arg, with an interval of 1 h between the injections. Subsequently, the rats were observed to assess whether hypothermia induced by 5'-AMP could effectively inhibit inflammation associated with L-Arg-induced acute pancreatitis in rats. Hypothermia induced by 5'-AMP produced protective effects in our acute pancreatitis model. These effects exhibited the following manifestations: (i) a significant reduction in rat mortality rates; (ii) a significant decrease in the occurrence of pancreatic edema; (iii) significant reductions in serum amylase (P < 0.001), interleukin-6 (P < 0.001), interleukin-1β (P < 0.001) and tumor necrosis factor-α (P < 0.001); (iv) the significant inhibition of nuclear factor-κB (NF-κB) activation in rats that were pre- and posttreated with 5'-AMP compared with rats that were only injected with L-Arg; and (v) significant decreases in the occurrence of pancreatic interstitial edema, inflammatory cell infiltration, hemorrhage, and acinar cell necrosis. Hypothermia induced by 5'-AMP could inhibit the acute inflammatory reaction and NF-κB activation associated with acute pancreatitis. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Staphylococcus aureus synthesizes adenosine to escape host immune responses

PubMed Central

Thammavongsa, Vilasack; Kern, Justin W.; Missiakas, Dominique M.

2009-01-01

Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses. PMID:19808256

Stepwise hydration and evaporation of adenosine monophosphate nucleotide anions: a multiscale theoretical study.

PubMed

Calvo, F; Douady, J

2010-04-14

The structure and finite-temperature properties of hydrated nucleotide anion adenosine 5'-monophosphate (AMP) have been theoretically investigated with a variety of methods. Using a polarizable version of the Amber force field and replica-exchange molecular dynamics simulations, putative lowest-energy structures have been located for the AMP(-)(H(2)O)(n) cluster anions with n = 0-20. The hydration energies obtained with the molecular mechanics potential slightly overestimate experimental measurements. However, closer values are found after reoptimizing the structures locally at more sophisticated levels, namely semi-empirical (PM6) and density-functional theory (B3LYP/6-31+G*). Upon heating the complexes, various indicators such as the heat capacity, number of hydrogen bonds or surface area provide evidence that the water cluster melts below 200 K but remains bonded to the AMP anion. The sequential loss of water molecules after sudden heating has been studied using a statistical approach in which unimolecular evaporation is described using the orbiting transition state version of phase space theory, together with anharmonic densities of vibrational states. The evaporation rates are calibrated based on the results of molecular dynamics trajectories at high internal energy. Our results indicate that between 4 and 10 water molecules are lost from AMP(-)(H(2)O)(20) after one second depending on the initial heating in the 250-350 K range, with a concomitant cooling of the remaining cluster by 75-150 K.

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Effect of parathyroid hormone and insulin on extracellular cyclic adenosine-3',5'-monophosphate in patients with benign and malignant breast tumors.

PubMed

Berstein, L M; Semiglazov, V F; Vishnevski, A S; Dilman, V M

1978-01-01

Basal excretion of cyclic adenosine monophosphate (cAMP) and its basal level in blood plasma in breast cancer (BC) patients and those with fibroadenomatosis did not differ essentially. However, intravenous injection of parathyroid hormone (100 U) and insulin (0.08 U/kg body weight) was followed by a much less rise in urine-cAMP excretion and blood-cAMP levels in BC patients than in benign process in mammary gland. A substantial correlation between changes in plasma cAMP level and the degree of insulin-induced hypoglycemia was not observed. There was a negative correlation between reponse to parathyroid hormone and insulin and body overweight in BC patients. It was suggested that body fat content may influence the peculiarities of metabolism of extracellular cAMP in cancer patients considerably.

The Effect of Endogenous Adenosine on Neuronal Activity in Rats: An FDG PET Study

PubMed Central

Paul, Soumen; Zhang, Dali; Mzengeza, Shadreck; Ko, Ji Hyun

2016-01-01

ABSTRACT 2–18F‐fluorodeoxy‐D‐glucose (FDG) is a glucose analog that is taken up by cells and phosphorylated. The amount of FDG accumulated by cells is a measure of the rate of glycolysis, which reflects cellular activity. As the levels and actions of the neuromodulator adenosine are dynamically regulated by neuronal activity, this study was designed to test whether endogenous adenosine affects tissue accumulation of FDG as assessed by positron emission tomography (PET) or by postmortem analysis of tissue radioactivity. Rats were given an intraperitoneal injection of the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropyl‐xanthine (DPCPX, 3 mg/kg), the adenosine kinase inhibitor ABT‐702 (3 mg/kg), or vehicle 10 minutes prior to an intravenous injection of FDG (15.4 ± 0.7 MBq per rat). Rats were then subjected to a 15 minute static PET scan. Reconstructed images were normalized to FDG PET template for rats and standard uptake values (SUVs) were calculated. To examine the regional effect of active treatment compared to vehicle, statistical parametric mapping analysis was performed. Whole‐brain FDG uptake was not affected by drug treatment. Significant regional hypometabolism was detected, particularly in cerebellum, of DPCPX‐ and ABT‐702 treated rats, relative to vehicle‐treated rats. Thus, endogenous adenosine can affect FDG accumulation although this effect is modest in quiescent rats. PMID:27082948

Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

PubMed

Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

2017-10-01

Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

Bacillus anthracis Edema Factor Substrate Specificity: Evidence for New Modes of Action

PubMed Central

Göttle, Martin; Dove, Stefan; Seifert, Roland

2012-01-01

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5′-triphosphate, uridine 5′-triphosphate and inosine 5′-triphosphate, in addition to adenosine 5′-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3′:5′-monophosphate, cyclic uridine 3′:5′-monophosphate and cyclic inosine 3′:5′-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed. PMID:22852066

THE SHARK RECTAL GLAND MODEL: A CHAMPION OF RECEPTOR MEDIATED CHLORIDE SECRETION THROUGH CFTR

PubMed Central

FORREST, JOHN N.

2016-01-01

The dogfish shark salt gland was predicted by Smith and discovered by Burger at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine. It is an epithelial organ in the intestine composed of tubules that serve a single function: the secretion of hypertonic NaCl. Many G protein receptors are present on the basolateral surface of these tubules, including stimulatory receptors for vasoactive intestinal peptide, adenosine A2, growth hormone releasing hormone, and inhibitory receptors for somatostatin and adenosine A1. An entirely different class of stimulatory receptors is present as C-type natriuretic peptide receptors. Each stimulatory receptor evokes powerful NaCl secretion. G protein receptors bind to Gαs to activate the catalytic unit of adenylate cyclase to form cyclic adenosine monophosphate (cAMP) and protein kinase A that phosphorylates the regulatory domain of cystic fibrosis transmembrane conductance regulator, opening the channel. The C-type natriuretic peptide receptor stimulates by activating guanylate cyclase and endogenous cyclic guanosine monophosphate which inhibits type 3 phosphodiesterase, the enzyme that breaks down cAMP, thereby elevating cAMP and activating the protein kinase A pathway. PMID:28066051

Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

PubMed

Yoshikawa, Noriko; Yamada, Shizuo; Takeuchi, Chihiro; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru; Nakamura, Kazuki

2008-06-01

Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor.

PubMed

Jarvis, Michael F; Yu, Haixia; McGaraughty, Steve; Wismer, Carol T; Mikusa, Joe; Zhu, Chang; Chu, Katharine; Kohlhaas, Kathy; Cowart, Marlon; Lee, Chih Hung; Stewart, Andrew O; Cox, Bryan F; Polakowski, James; Kowaluk, Elizabeth A

2002-03-01

Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO-metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti-inflammatory agents. N7-((1'R,2'S,3'R,4'S)-2',3'-dihydroxy-4'-amino-cyclopentyl)-4-amino-5-bromo-pyrrolo[2,3-a]pyrimidine (A-286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50 >100 microM) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 value >10 microM) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases-1 and -2. A-286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A-286501 was a reversible and competitive inhibitor with respect to ADO and non-competitive with respect to MgATP2-. A-286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin-induced diabetic) pain. A-286501 was particularly potent (ED50=1 micromol/kg, p.o.) to reduce carrageenan-induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A-286501 was also effective to reduce carrageenan-induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 micromol/kg, p.o.), in the injured paw. The anti-nociceptive effects of A-286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 micromol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following

Intracellular ATP influences synaptic plasticity in area CA1 of rat hippocampus via metabolism to adenosine and activity-dependent activation of adenosine A1 receptors.

PubMed

zur Nedden, Stephanie; Hawley, Simon; Pentland, Naomi; Hardie, D Grahame; Doney, Alexander S; Frenguelli, Bruno G

2011-04-20

The extent to which brain slices reflect the energetic status of the in vivo brain has been a subject of debate. We addressed this issue to investigate the recovery of energetic parameters and adenine nucleotides in rat hippocampal slices and the influence this has on synaptic transmission and plasticity. We show that, although adenine nucleotide levels recover appreciably within 10 min of incubation, it takes 3 h for a full recovery of the energy charge (to ≥ 0.93) and that incubation of brain slices at 34°C results in a significantly higher ATP/AMP ratio and a threefold lower activity of AMP-activated protein kinase compared with slices incubated at room temperature. Supplementation of artificial CSF with d-ribose and adenine (Rib/Ade) increased the total adenine nucleotide pool of brain slices, which, when corrected for the influence of the dead cut edges, closely approached in vivo values. Rib/Ade did not affect basal synaptic transmission or paired-pulse facilitation but did inhibit long-term potentiation (LTP) induced by tetanic or weak theta-burst stimulation. This decrease in LTP was reversed by strong theta-burst stimulation or antagonizing the inhibitory adenosine A(1) receptor suggesting that the elevated tissue ATP levels had resulted in greater activity-dependent adenosine release during LTP induction. This was confirmed by direct measurement of adenosine release with adenosine biosensors. These observations provide new insight into the recovery of adenine nucleotides after slice preparation, the sources of loss of such compounds in brain slices, the means by which to restore them, and the functional consequences of doing so.

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma X glioma hybrid cells.

PubMed Central

Snell, C. R.; Snell, P. H.

1984-01-01

We have demonstrated high affinity diazepam binding sites of the Ro5-4864 benzodiazepine receptor subtype on 108CC15 neuroblastoma X glioma hybrid cells. These cells were previously shown to have purinoceptors of the A2 adenosine subtype and we have now found that [3H]-adenosine can be displaced from this binding site by the benzodiazepines and related compounds that can also bind to the Ro5-4864 site. Diazepam was found to have no intrinsic activity at the A2-receptor as measured by the stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in this cell line. At concentrations sufficient to compete for the A2-receptor, diazepam was shown to facilitate, by approximately 2 fold, the stimulation of cyclic AMP by adenosine. These effects are not due to inhibition of adenosine uptake or phosphodiesterase activity, but are probably a consequence of modulation of the coupling of the A2-receptor to cyclic AMP production in this hybrid cell line. PMID:6150742

The effect of adenosine 5'-monophosphate (AMP) on tenderness, microstructure and chemical-physical index of duck breast meat.

PubMed

Wang, Daoying; Deng, Shaoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

2016-03-30

Adenosine 5'-monophosphate (AMP) is often used in meat and poultry soups as a flavor enhancer (flavor modifier), or as food additives for specific nutritional purposes. Our previous research as well as evidence from others showed that actomyosin could be dissociated into myosin and actin by AMP in extracted muscle solution. However, there is no report available on the application of AMP to dissociate actomyosin and to improve meat tenderness. The objectives of this study were to evaluate the effect of AMP on duck meat tenderness and other quality traits and to explore the mechanism of the action of AMP on meat tenderness. Duck breast muscle was treated with 0, 10, 20, 30, 40 mmol L(-1) AMP at 5 °C for 10 h and examined for shear force, microstructure, actomyosin dissociation, myofibril fragmentation index (MFI), pH, water content, cooking loss, CIE* color (L*, a*, b*), inosine monophosphate (IMP) and free amino acid (FAA) contents. Results showed that shear force, cooking loss, L* and b* of the muscles significantly decreased after AMP treatment (P < 0.05); actomyosin dissociation, MFI, pH, water content, fiber diameter, sarcomere length, IMP and ammonia significantly increased (P < 0.05); no significant change in a* or other FAA content was observed (P > 0.05), and muscle shrinkage in transverse and longitudinal directions were restrained after AMP treatment. The results suggest that AMP could notably improve meat tenderness, and this effect was probably mainly through increasing muscle pH, promoting actomyosin dissociation and disrupting the Z-line; meanwhile, the conversion of AMP to IMP may contribute to the flavor of meat. © 2015 Society of Chemical Industry.

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

PubMed

Gross, W; Yang, W; Boss, W F

1992-02-19

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

AMP-activated protein kinase confers protection against TNF-{alpha}-induced cardiac cell death.

PubMed

Kewalramani, Girish; Puthanveetil, Prasanth; Wang, Fang; Kim, Min Suk; Deppe, Sylvia; Abrahani, Ashraf; Luciani, Dan S; Johnson, James D; Rodrigues, Brian

2009-10-01

Although a substantial role for 5' adenosine monophosphate-activated protein kinase (AMPK) has been established in regulating cardiac metabolism, a less studied action of AMPK is its ability to prevent cardiac cell death. Using established AMPK activators like dexamethasone (DEX) or metformin (MET), the objective of the present study was to determine whether AMPK activation prevents tumour necrosis factor-alpha (TNF-alpha) induced apoptosis in adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with DEX, MET, or TNF-alpha for varying durations (0-12 h). TNF-alpha-induced cell damage was evaluated by measuring caspase-3 activity and Hoechst staining. Protein and gene estimation techniques were employed to determine the mechanisms mediating the effects of AMPK activators on TNF-alpha-induced cardiomyocyte apoptosis. Incubation of myocytes with TNF-alpha for 8 h has increased caspase-3 activation and apoptotic cell death, an effect that was abrogated by DEX and MET. The beneficial effect of DEX and MET was associated with stimulation of AMPK, which led to a rapid and sustained increase in Bad phosphorylation. This event reduced the interaction between Bad and Bcl-xL, limiting cytochrome c release and caspase-3 activation. Addition of Compound C to inhibit AMPK reduced Bad phosphorylation and prevented the beneficial effects of AMPK against TNF-alpha-induced cytotoxicity. Our data demonstrate that although DEX and MET are used as anti-inflammatory agents or insulin sensitizers, respectively, their common property to phosphorylate AMPK promotes cardiomyocyte cell survival through its regulation of Bad and the mitochondrial apoptotic mechanism.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis*

PubMed Central

Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E.; Rhee, Kyu Y.; Jacobs, William R.; Berney, Michael; Blanchard, John S.

2016-01-01

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests that Mtb relies mainly on fatty acid catabolism in the host. However, Mtb also maintains a functional glycolytic pathway and its role in the cellular metabolism of Mtb has yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and the Mtb genome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show that pykA encodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion of pykA prevents Mtb growth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism in Mtb. PMID:26858255

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

PubMed Central

Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

2013-01-01

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

PubMed Central

Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

2015-01-01

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

Innate Immune Response to Burkholderia mallei

DTIC Science & Technology

2017-02-16

stimulate immune responses via TLR4 activation that may contribute to persistent infection. Summary Mortality is high due to septicemia and immune...phosphorylation of adenosine monophosphate- activated protein kinase (AMPK); regulators of NF-κB signaling pathway (e.g. IκBα, GSK3β, Src, and STAT1) and mitogen... activated protein kinases (e.g. p38, ERK1/2 and c-Myc) (13). The degrees in which target host proteins or processes are modulated correlated to the

Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

PubMed Central

Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

2014-01-01

Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

Liver X receptor agonist alleviated high glucose-induced endothelial progenitor cell dysfunction via inhibition of reactive oxygen species and activation of AMP-activated protein kinase.

PubMed

Li, Xiaoxia; Song, Yimeng; Han, Yingying; Wang, Dawei; Zhu, Yi

2012-08-01

Liver X receptors (LXRs) are key regulators of cholesterol homeostasis. Synthetic LXR agonists are anti-atherogenic and anti-inflammatory. However, the effect of LXR agonists on endothelial progenitor cell (EPC) function is largely unknown. Here, we explored the effect of the LXR agonist TO901317 (TO) on EPC biology and the underlying mechanisms. Endothelial progenitor cells were cultured in mannitol or 30 mm glucose (high glucose) for 24 hours. For TO treatments, cells were pretreated with TO (10 μm) for 12 hours, then mannitol or high glucose was added for an additional 24 hours. EPCs function, reactive oxygen species (ROS) release, and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) were analyzed. TO could restore the high glucose-impaired adhesion and migration capacity of EPCs. High glucose impaired EPC-mediated angiogenesis, and TO reversed the impairment. TO also alleviated ROS release induced by high glucose. Western blot analysis revealed that high glucose downregulated the phosphorylation of AMPK and endothelial nitric oxide synthase, which could be reversed with TO treatment. Furthermore, inhibiting AMPK activation by compound C could abolish the protective effects of TO on EPCs. TO had a protective effect on EPCs under high glucose by inhibiting ROS release and activating AMPK. © 2012 John Wiley & Sons Ltd.

Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

2010-09-01

Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results:more » IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.« less

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

PubMed

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

PubMed

Young, Li-Sen; Harrison, Benjamin R; Narayana Murthy, U M; Moffatt, Barbara A; Gilroy, Simon; Masson, Patrick H

2006-10-01

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

PubMed

Wei, C-B; Wang, J-Q; Chen, F-Y; Niu, H; Li, K

2015-02-06

The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

[High performance liquid chromatogram (HPLC) determination of adenosine phosphates in rat myocardium].

PubMed

Miao, Yu; Wang, Cheng-long; Yin, Hui-jun; Shi, Da-zhuo; Chen, Ke-ji

2005-04-18

To establish method for the quantitative determination of adenosine phosphates in rat myocardium by optimized high performance liquid chromatogram (HPLC). ODS HYPERSIL C(18) column and a mobile phase of 50 mmol/L tribasic potassium phosphate buffer solution (pH 6.5), with UV detector at 254 nm were used. The average recovery rates of myocardial adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were 99%-107%, 96%-104% and 95%-119%, respectively; relative standard deviations (RSDs) of within-day and between-days were less than 1.5% and 5.1%, respectively. The method is simple, rapid and accurate, and can be used to analyse the adenosine phosphates in myocardium.

Houttuynia cordata attenuates lipid accumulation via activation of AMP-activated protein kinase signaling pathway in HepG2 cells.

PubMed

Kang, Hyun; Koppula, Sushruta

2014-01-01

Houttuynia cordata (H. cordata) from the family Saururaceae is a perennial herb native to Southeast Asia. It possesses a range of medicinal properties to treat several disease symptoms including allergic inflammation and anaphylaxis. In the present investigation, we provided the molecular mechanisms underlying the role of H. cordata extract (HCE) in the prevention of high glucose-induced lipid accumulation in human HepG2 hepatocytes. HepG2 cells were pre-treated with various concentrations of HCE (0, 10, 20, 40, and 80 μg/mL) and treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (25 mM D-glucose) for 24 h. HCE significantly and dose-dependently attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (25 mM D-glucose) (p < 0.05, p < 0.01 and p < 0.001 at 20, 40, and 80 μg/mL concentrations, respectively). Further, HCE attenuated the expression of fatty acid synthase (FAS), sterol regulatory element-binding protein-1 and glycerol 3-phosphate acyltransferases (GPATs). The adenosine monophosphate-activated protein kinase (AMPK) was also activated by HCE treatment when exposed to high glucose (25 mM D-glucose) in human HepG2 hepatocytes. This study suggests the hypolipidemic effects of HCE by the inhibition of lipid biosynthesis mediated through AMPK signaling, which may play an active role and can be developed as an anti-obesity agent.

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana.

PubMed

Pereira, L A R; Schoor, S; Goubet, F; Dupree, P; Moffatt, B A

2006-11-01

Pectin methyl-esterification is catalysed by S-adenosyl-L: -methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.

Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

PubMed

Ross, Christina L; Teli, Thaleia; Harrison, Benjamin S

2016-01-01

During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Adenosine and the Auditory System

PubMed Central

Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R

2009-01-01

Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea. PMID:20190966

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

PubMed Central

Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

2015-01-01

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

Cellular Energetic Status Supervises the Synthesis of Bis-Diphosphoinositol Tetrakisphosphate Independently of AMP-Activated Protein Kinase

PubMed Central

Choi, Kuicheon; Mollapour, Elahe; Choi, Jae H.; Shears, Stephen B.

2009-01-01

Cells aggressively defend adenosine nucleotide homeostasis; intracellular biosensors detect variations in energetic status and communicate with other cellular networks to initiate adaptive responses. Here, we demonstrate some new elements of this communication process, and we show that this networking is compromised by off-target, bioenergetic effects of some popular pharmacological tools. Treatment of cells with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), so as to simulate elevated AMP levels, reduced the synthesis of bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4), an intracellular signal that phosphorylates proteins in a kinase-independent reaction. This was a selective effect; levels of other inositol phosphates were unaffected by AICAR. By genetically manipulating cellular AMP-activated protein kinase activity, we showed that it did not mediate these effects of AICAR. Instead, we conclude that the simulation of deteriorating adenosine nucleotide balance itself inhibited [PP]2-InsP4 synthesis. This conclusion is consistent with our demonstrating that oligomycin elevated cellular [AMP] and selectively inhibited [PP]2-InsP4 synthesis without affecting other inositol phosphates. In addition, we report that the short-term increases in [PP]2-InsP4 levels normally seen during hyperosmotic stress were attenuated by 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (PD184352). The latter is typically considered an exquisitely specific mitogen-activated protein kinase kinase (MEK) inhibitor, but small interfering RNA against MEK or extracellular signal-regulated kinase revealed that this mitogen-activated protein kinase pathway was not involved. Instead, we demonstrate that [PP]2-InsP4 synthesis was inhibited by PD184352 through its nonspecific effects on cellular energy balance. Two other MEK inhibitors, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) and 2′-amino-3′-methoxyflavone (PD98059), had similar off

Mechanisms of protein kinase C signaling in the modulation of 3',5'-cyclic adenosine monophosphate-mediated steroidogenesis in mouse gonadal cells.

PubMed

Manna, Pulak R; Huhtaniemi, Ilpo T; Stocco, Douglas M

2009-07-01

The protein kinase C (PKC) signaling pathway plays integral roles in the expression of the steroidogenic acute regulatory (StAR) protein that regulates steroid biosynthesis in steroidogenic cells. PKC can modulate the activity of cAMP/protein kinase A signaling involved in steroidogenesis; however, its mechanism remains obscure. In the present study, we demonstrate that activation of the PKC pathway, by phorbol 12-myristate 13-acetate (PMA), was capable of potentiating dibutyryl cAMP [(Bu)(2)cAMP]-stimulated StAR expression, StAR phosphorylation, and progesterone synthesis in both mouse Leydig (MA-10) and granulosa (KK-1) tumor cells. The steroidogenic potential of PMA and (Bu)(2)cAMP was linked with phosphorylation of ERK 1/2; however, inhibition of the latter demonstrated varying effects on steroidogenesis. Transcriptional activation of the StAR gene by PMA and (Bu)(2)cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of the cAMP response element binding protein (CREB). An oligonucleotide probe containing a CREB/activating transcription factor binding region in the StAR promoter was found to bind nuclear proteins in PMA and (Bu)(2)cAMP-treated MA-10 and KK-1 cells. Chromatin immunoprecipitation studies revealed that the induction of phosphorylated CREB was tightly correlated with in vivo protein-DNA interactions and recruitment of CREB binding protein to the StAR promoter. Ectopic expression of CREB binding protein enhanced CREB-mediated transcription of the StAR gene, an event that was markedly repressed by the adenovirus E1A oncoprotein. Further studies demonstrated that the activation of StAR expression and steroid synthesis by PMA and (Bu)(2)cAMP was associated with expression of the nuclear receptor Nur77, indicating its essential role in hormone-regulated steroidogenesis. Collectively, these findings provide insight into the mechanisms by which PKC modulates cAMP/protein kinase A responsiveness involved in

The prostaglandin receptor EP2 activates multiple signaling pathways and β-arrestin1 complex formation during mouse skin papilloma development

PubMed Central

Chun, Kyung-Soo; Lao, Huei-Chen; Trempus, Carol S.; Okada, Manabu; Langenbach, Robert

2009-01-01

Prostaglandin E2 (PGE2) is elevated in many tumor types, but PGE2's contributions to tumor growth are largely unknown. To investigate PGE2's roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors—cyclic adenosine 3′,5′-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2—were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE2 production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3′,5′-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2−/− mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR–β-arrestin–Src complex. Indeed, immunoprecipitation of β-arrestin1 or p-Src indicated the presence of an EP2–β-arrestin1–p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with β-arrestin1 and Src that contributed to signaling and/or EP2

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

PubMed Central

Zylka, Mark J.; Sowa, Nathaniel A.; Taylor-Blake, Bonnie; Twomey, Margaret A.; Herrala, Annakaisa; Voikar, Vootele; Vihko, Pirkko

2008-01-01

SUMMARY Thiamine monophosphatase (TMPase, also known as Fluoride-Resistant Acid Phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of Prostatic Acid Phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent anti-nociceptive, anti-hyperalgesic and anti-allodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5’-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. PMID:18940592

Circadian variations of adenosine and of its metabolism. Could adenosine be a molecular oscillator for circadian rhythms?

PubMed

Chagoya de Sánchez, V

1995-03-01

The present review describes the biological implications of the periodic changes of adenosine concentrations in different tissues of the rat. Adenosine is a purine molecule that could have been formed in the prebiotic chemical evolution and has been preserved. The rhythmicity of this molecule, as well as its metabolism and even the presence of specific receptors, suggests a regulatory role in eukaryotic cells and in multicellular organisms. Adenosine may be considered a chemical messenger and its action could take place at the level of the same cell (autocrine), the same tissue (paracrine), or on separate organs (endocrine). Exploration of the circadian variations of adenosine was planned considering the liver as an important tissue for purine formation, the blood as a vehicle among tissues, and the brain as the possible acceptor for hepatic adenosine or its metabolites. The rats used in these studies were adapted to a dark-light cycle of 12 h with an unrestrained feeding and drinking schedule. The metabolic control of adenosine concentration in the different tissues studied through the 24-h cycle is related to the activity of adenosine-metabolizing enzyme: 5'-nucleotidase adenosine deaminase, adenosine kinase, and S-adenosylhomocysteine hydrolase. Some possibilities of the factors modulating the activity of these enzymes are commented upon. The multiphysiological action of adenosine could be mediated by several actions: (i) by interaction with extracellular and intracellular receptors and (ii) through its metabolism modulating the methylation pathway, possibly inducing physiological lipoperoxidation, or participating in the energetic homeostasis of the cell. The physiological meaning of the circadian variations of adenosine and its metabolism was focused on: maintenance of the energetic homeostasis of the tissues, modulation of membrane structure and function, regulation of fasting and feeding metabolic pattern, and its participation in the sleep-wake cycle. From

Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors.

PubMed

Yamaguchi, Momoka; Saito, Shin-Ya; Nishiyama, Ryota; Nakamura, Misuzu; Todoroki, Kenichiro; Toyo'oka, Toshimasa; Ishikawa, Tomohisa

2017-01-01

During liver injury, hepatic stellate cells (HSCs) are activated by various cytokines and transdifferentiated into myofibroblast-like activated HSCs, which produce collagen, a major source of liver fibrosis. Therefore, the suppression of HSC activation is regarded as a therapeutic target for liver fibrosis. Several epidemiological reports have revealed that caffeine intake decreases the risk of liver disease. In this study, therefore, we investigated the effect of caffeine on the activation of primary HSCs isolated from mice. Caffeine suppressed the activation of HSC in a concentration-dependent manner. BAPTA-AM, an intracellular Ca 2+ chelator, had no effect on the caffeine-induced suppression of HSC activation. None of the isoform-selective inhibitors of phosphodiesterase1 to 5 affected changes in the morphology of HSC during activation, whereas CGS-15943, an adenosine receptor antagonist, inhibited them. Caffeine had no effect on intracellular cAMP level or on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. In contrast, caffeine significantly decreased the phosphorylation of Akt1. These results suggest that caffeine inhibits HSC activation by antagonizing adenosine receptors, leading to Akt1 signaling activation.

A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

PubMed Central

2012-01-01

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription. PMID:22273182

Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

PubMed Central

Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

2012-01-01

Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

MRI and (1)H-MRS in adenosine kinase deficiency.

PubMed

Staufner, C; Blom, H J; Dionisi-Vici, C; Freisinger, P; Makhseed, N; Ballhausen, D; Kölker, S; Hoffmann, G F; Harting, I

2016-07-01

Adenosine kinase deficiency (ADK deficiency) is a recently described disorder of methionine and adenosine metabolism resulting in a neurological phenotype with developmental delay, muscular hypotonia, and epilepsy as well as variable systemic manifestations. The underlying neuropathology is poorly understood. We have investigated MRI and (1)H-MRS changes in ADK deficiency in order to better understand the in vivo neuropathologic changes of ADK deficiency. Systematic evaluation of 21 MRIs from eight patients (age range 9 days-14.6 years, mean 3.9 years, median 2.7 years) including diffusion-weighted imaging in six and (1)H-MRS in five patients. Brain maturation was delayed in the neonatal period and in infancy (6/6), but ultimately complete. White matter changes occurring in five of eight patients were discrete, periventricular, and unspecific (4/5), or diffuse with sparing of optic radiation, corona radiata, and pyramidal tracts (1/5). Choline was low in white matter spectra (3/3), while there was no indication of low creatine in white matter or basal ganglia (5/5), and diffusion was variably decreased or increased. Central tegmental tract hyperintensity was a common finding (6/8), as was supratentorial atrophy (6/8). MRI changes in ADK deficiency consist of delayed but ultimately completed brain maturation with later onset of mostly unspecific white matter changes and potentially transient central tegmental tract hyperintensity. Immaturity on neonatal MRI is consistent with prenatal onset of disease and reduced choline with lower membrane turnover resulting in delayed myelination and deficient myelin maintenance.

Adenosine-derived doped carbon dots: From an insight into effect of N/P co-doping on emission to highly sensitive picric acid sensing.

PubMed

Li, Na; Liu, Shi Gang; Fan, Yu Zhu; Ju, Yan Jun; Xiao, Na; Luo, Hong Qun; Li, Nian Bing

2018-07-12

The various synthetic routes of carbon dots (C-dots) feature a considerable step toward their potential use in chemical sensors and biotechnology. Herein, by coupling phosphorus and nitrogen element introduction, the adenosine-derived N/P co-doped C-dots with fluorescence enhancement were achieved. By separately employing adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine-5'-triphosphate as precursors, the effect of N/P co-doping on the fluorescence emission is discussed in detail. The formed C-dots with adenosine monophosphate exhibited strong blue fluorescence with a high quantum yield of 33.81%. Then the C-dots were employed as a fluorescent probe and utilized to develop a fast, sensitive, and selective picric acid sensor. The fluorescence of C-dots can be quenched by picric acid immediately, giving rise to a picric acid determination down to 30 nM. The possible mechanism of fluorescence quenching was discussed, which was proved to be inner filter effect and static quenching. Moreover, this method has the potential to detect picric acid in environmental water samples. Copyright © 2018 Elsevier B.V. All rights reserved.

5'-adenosine monophosphate-induced hypothermia attenuates brain ischemia/reperfusion injury in a rat model by inhibiting the inflammatory response.

PubMed

Miao, Yi-Feng; Wu, Hui; Yang, Shao-Feng; Dai, Jiong; Qiu, Yong-Ming; Tao, Zhen-Yi; Zhang, Xiao-Hua

2015-01-01

Hypothermia treatment is a promising therapeutic strategy for brain injury. We previously demonstrated that 5'-adenosine monophosphate (5'-AMP), a ribonucleic acid nucleotide, produces reversible deep hypothermia in rats when the ambient temperature is appropriately controlled. Thus, we hypothesized that 5'-AMP-induced hypothermia (AIH) may attenuate brain ischemia/reperfusion injury. Transient cerebral ischemia was induced by using the middle cerebral artery occlusion (MCAO) model in rats. Rats that underwent AIH treatment exhibited a significant reduction in neutrophil elastase infiltration into neuronal cells and matrix metalloproteinase 9 (MMP-9), interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), and Toll-like receptor (TLR) protein expression in the infarcted area compared to euthermic controls. AIH treatment also decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive neuronal cells. The overall infarct volume was significantly smaller in AIH-treated rats, and neurological function was improved. By contrast, rats with ischemic brain injury that were administered 5'-AMP without inducing hypothermia had ischemia/reperfusion injuries similar to those in euthermic controls. Thus, the neuroprotective effects of AIH were primarily related to hypothermia.

Thyrotropin-induced hydrogen peroxide production in FRTL-5 thyroid cells is mediated not by adenosine 3',5'-monophosphate, but by Ca2+ signaling followed by phospholipase-A2 activation and potentiated by an adenosine derivative.

PubMed

Kimura, T; Okajima, F; Sho, K; Kobayashi, I; Kondo, Y

1995-01-01

The production of hydrogen peroxide (H2O2) as an essential process for iodide organification is a key reaction in TSH-induced thyroid hormone synthesis. Here we characterize the signal transduction pathway involved in TSH-induced H2O2 production in FRTL-5 thyroid cells. At higher than 1 nM TSH, N6-(L-2-phenylisopropyl)adenosine (PIA), an adenosine receptor agonist having, by itself, no influence on H2O2 generation, potentiated this TSH action, whereas the TSH increase and PIA addition reduced cAMP accumulation. RO 20-1724, a phosphodiesterase inhibitor, amplified the TSH-induced cAMP accumulation, but did not change H2O2 generation in the whole range of TSH used. Ca(2+)-mobilizing agonists, GTP and ATP, also induced H2O2 production without stimulating cAMP accumulation. Chelation of intracellular Ca2+ markedly inhibited the TSH action, but intracellular Ca2+ increases by either thapsigargin or ionomycin mimicking it. All of the findings show the participation of Ca2+, but not cAMP, in the action of TSH. Desensitization of protein kinase-C (PKC) did not influence the receptor-mediated H2O2 production, suggesting the reduced importance of PKC activation compared to Ca2+ signaling to the reaction. A rise in intracellular Ca2+ independent of receptor activation also induced H2O2 production as well as arachidonate release, and both were potentiated by PIA. In addition, inhibitors of phospholipase-A2 and the arachidonate metabolic pathway depressed H2O2 generation, suggesting the participation of an arachidonate cascade in the Ca(2+)-dependent H2O2 production. Lipoxygenase inhibitors depressed the Ca2+ action without influencing arachidonate release, suggesting the involvement of a lipoxygenase product(s) of arachidonate in the Ca(2+)-signaling mechanism. In conclusion, in FRTL-5 cells, TSH-induced H2O2 production is mediated not by cAMP, but by the phospholipase-C/Ca2+ cascade, possibly followed by the Ca(2+)-dependent phospholipase-A2/arachidonate cascade. PIA

The Clinical Correlation of Regulatory T Cells and Cyclic Adenosine Monophosphate in Enterovirus 71 Infection

PubMed Central

Wang, Shih-Min; Chen, I-Chun; Liao, Yu-Ting; Liu, Ching-Chuan

2014-01-01

Background Brainstem encephalitis (BE) and pulmonary edema (PE) are notable complications of enterovirus 71 (EV71) infection. Objective This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment. Study Design Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD) or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP) levels, and the regulatory T cell (Tregs) profiles of the patients were determined. Results Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4+CD25+Foxp3+ and CD4+Foxp3+ T cells compared with patients with HFMD or BE. The expression frequency of CD4−CD8− was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4+Foxp3+ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment. Conclusions These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels. PMID:25010330

Cyclic Adenosine Monophosphate Accumulation and beta-Adrenergic Binding in Unweighted and Denervated Rat Soleus Muscle

NASA Technical Reports Server (NTRS)

Kirby, Christopher R.; Woodman, Christopher R.; Woolridge, Dale; Tischler, Marc E.

1992-01-01

Unweighting, but not denervation, of muscle reportedly "spares" insulin receptors, increasing insulin sensitivity. Unweighting also increases beta-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and beta-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a p-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular (IM) injection) to a greater degree (P less than .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased beta-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B(sub max)) for iodo-125(-)-pindolol in particulate preparations of unweighted (420 x 10(exp -18) mol/mg muscle) than of control or denervated muscles (285 x 10(exp-18) mol/mg muscle). The three dissociation constant (Kd) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the "sparing" and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished CAMP accumulation in the presence of theophylline in unweighted muscle (-66% +/- 2%) more than in controls (-42% +'- 6%, P less than .001). These results show that insulin affects CAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

RNA Initiation with Dinucleoside Monophosphates during Transcription of Bacteriophage T4 DNA with RNA Polymerase of Escherichia coli

PubMed Central

Hoffman, David J.; Niyogi, Salil K.

1973-01-01

The effects of dinucleoside monophosphates on the transcription of phage T4 DNA by E. coli RNA polymerase have been examined at various concentrations of the sigma subunit and extremely low concentration of ribonucleoside triphosphate. The following conclusions were reached: (i) Labeled specific dinucleoside monophosphates are incorporated as chain initiators. (ii) When the ratio of sigma factor to core enzyme is small, there is a general stimulation by most 5′-guanosyl dinucleoside monophosphates. (iii) When the ratio is increased or holoenzyme is present, ApU, CpA, UpA, and GpU are the most effective stimulators. (iv) At high concentrations of sigma factor, only certain adenosine-containing dinucleoside monophosphates (ApU, CpA, UpA, and ApA) stimulate the reaction. (v) Competition hybridization studies indicate that the RNAs stimulated by dinucleoside monophosphates (ApU, CpA, UpA, and GpU) are of the T4 “early” type. (vi) Studies involving both combinations of stimulatory dinucleoside monophosphates and competitive effects of these compounds on chain initiation by ATP and GTP suggest that the stimulatory dinucleoside monophosphates act as chain initiators and may recognize part of a continuous sequence in a promoter region. Studies based on the incorporation of 3H-labeled stimulatory dinucleoside monophosphates support the above conclusions. PMID:4568732

5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers.

PubMed Central

Madara, J L; Patapoff, T W; Gillece-Castro, B; Colgan, S P; Parkos, C A; Delp, C; Mrsny, R J

1993-01-01

Neutrophil transmigration across intestinal epithelia is thought to contribute to epithelial dysfunction and characterizes many inflammatory intestinal diseases. Neutrophils activated by factors, normally present in the lumen, release a neutrophil-derived secretagogue activity to which intestinal epithelia respond with an electrogenic chloride secretion, the transport event which underlies secretory diarrhea. Using sequential ultrafiltration, column chromatographic, and mass and Raman spectroscopic techniques, neutrophil-derived secretagogue was identified as 5'-AMP. Additional studies suggested that neutrophil-derived 5'-AMP is subsequently converted to adenosine at the epithelial cell surface by ecto-5'-nucleotidase and that adenosine subsequently activates intestinal secretion through adenosine receptors on the apical membrane of target intestinal epithelial cells. These findings suggest that this ATP metabolite may serve as a neutrophil-derived paracrine mediator that contributes to secretory diarrhea in states of intestinal inflammation. PMID:8486793

Application and optimization of the tenderization of pig Longissimus dorsi muscle by adenosine 5'-monophosphate (AMP) using the response surface methodology.

PubMed

Deng, Shaoying; Wang, Daoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

2016-03-01

Based on single factor experiments, NaCl concentration, adenosine 5'-monophosphate (AMP) concentration and temperature were selected as independent variables for a three-level Box-Behnken experimental design, and the shear force and cooking loss were response values for regression analysis. According to the statistical models, it showed that all independent variables had significant effects on shear force and cooking loss, and optimal values were at the NaCl concentration of 4.15%, AMP concentration of 22.27 mmol/L and temperature of 16.70°C, which was determined with three-dimensional response surface diagrams and contour plots. Under this condition, the observed shear force and cooking loss were 0.625 kg and 8.07%, respectively, exhibiting a good agreement with their predicted values, showing the good applicability and feasibility of response surface methodology (RSM) for improving pork tenderness. Compared with control pig muscles, AMP combined with NaCl treatment demonstrated significant effects on improvement of meat tenderness and reduction of cooking loss. Therefore, AMP could be regarded as an effective tenderization agent for pork. © 2015 Japanese Society of Animal Science.

Rapid activation by 3,5,3'-L-triiodothyronine of adenosine 5'-monophosphate-activated protein kinase/acetyl-coenzyme a carboxylase and akt/protein kinase B signaling pathways: relation to changes in fuel metabolism and myosin heavy-chain protein content in rat gastrocnemius muscle in vivo.

PubMed

de Lange, Pieter; Senese, Rosalba; Cioffi, Federica; Moreno, Maria; Lombardi, Assunta; Silvestri, Elena; Goglia, Fernando; Lanni, Antonia

2008-12-01

T3 stimulates metabolic rate in many tissues and induces changes in fuel use. The pathways by which T3 induces metabolic/structural changes related to altered fuel use in skeletal muscle have not been fully clarified. Gastrocnemius muscle (isolated at different time points after a single injection of T3 into hypothyroid rats), displayed rapid inductions of AMP-activated protein kinase (AMPK) phosphorylation (threonine 172; within 6 h) and acetyl-coenzyme A carboxylase phosphorylation (serine 79; within 12 h). As a consequence, increases occurred in mitochondrial fatty acid oxidation and carnitine palmitoyl transferase activity. Concomitantly, T3 stimulated signaling toward increased glycolysis through a rapid increase in Akt/protein kinase B (serine 473) phosphorylation (within 6 h) and a directly related increase in the activity of phosphofructokinase. The kinase specificity of the above effects was verified by treatment with inhibitors of AMPK and Akt activity (compound C and wortmannin, respectively). In contrast, glucose transporter 4 translocation to the membrane (activated by T3 within 6 h) was maintained when either AMPK or Akt activity was inhibited. The metabolic changes were accompanied by a decline in myosin heavy-chain Ib protein [causing a shift toward the fast-twitch (glycolytic) phenotype]. The increases in AMPK and acetyl-coenzyme A carboxylase phosphorylation were transient events, both levels declining from 12 h after the T3 injection, but Akt phosphorylation remained elevated until at least 48h after the injection. These data show that in skeletal muscle, T3 stimulates both fatty acid and glucose metabolism through rapid activations of the associated signaling pathways involving AMPK and Akt/protein kinase B.

Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

NASA Technical Reports Server (NTRS)

Prabahar, K. J.; Ferris, J. P.

1997-01-01

Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

[Effects of dopamine and adenosine on regulation of water-electrolyte exchange in Amoeba proteus].

PubMed

Bagrov, Ia Iu; Manusova, N B

2014-01-01

Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms.

Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

PubMed

Mino, Yasuaki; Naito, Takafumi; Shimoyama, Kumiko; Ogawa, Noriyoshi; Kawakami, Junichi

2017-07-01

Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7  µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R 2  = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase

Relaxation effect of abacavir on rat basilar arteries.

PubMed

Li, Rachel Wai Sum; Yang, Cui; Chan, Shun Wan; Hoi, Maggie Pui Man; Lee, Simon Ming Yuen; Kwan, Yiu Wa; Leung, George Pak Heng

2015-01-01

The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels. The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5' nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate. Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5' nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries. Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to endothelial dysfunction

Evidence for the role of hydrophobic forces on the interactions of nucleotide-monophosphates with cationic liposomes.

PubMed

Cuomo, Francesca; Mosca, Monica; Murgia, Sergio; Avino, Pasquale; Ceglie, Andrea; Lopez, Francesco

2013-11-15

In this work, the interaction of nucleotide-monophosphates (NMPs) with unilamellar liposomes made of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) was investigated. Here, we demonstrate how adsorption is affected by the type of nucleotide-monophosphate. Dynamic light scattering (DLS) results revealed, for each NMP, that a distinguishable concentration exists at which a significant growth of the aggregates occurs. Adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) have shown a higher propensity to induce liposome aggregation process and in particular GMP appears to be the most effective. From ζ-potential experiments we found that liposomes loaded with purine based nucleotides (AMP and GMP) are able to decrease the ζ-potential values to a greater extent in comparison with the pyrimidine based nucleotides thimydine 5'-monophosphate (TMP) and uridine 5'-monophosphate (UMP). Moreover, a careful analysis of nucleotide-liposome interactions revealed that nucleotides have different capacity to induce the formation of nucleotide-liposome complexes, and purine based nucleotides have higher affinities with lipid membranes. On the whole, the data emphasize that the mechanisms driving the interactions between liposomes and NMPs are also influenced by the existence of hydrophobic forces. Copyright © 2013 Elsevier Inc. All rights reserved.

Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

PubMed

Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

2015-07-01

4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Hydro-ethanolic extract of cashew tree (Anacardium occidentale) nut and its principal compound, anacardic acid, stimulate glucose uptake in C2C12 muscle cells.

PubMed

Tedong, Leonard; Madiraju, Padma; Martineau, Louis C; Vallerand, Diane; Arnason, John T; Desire, Dzeufiet D P; Lavoie, Louis; Kamtchouing, Pierre; Haddad, Pierre S

2010-12-01

Products of cashew tree (Anacardium occidentale) are used in traditional medicine for various ailments, including diabetes. The anti-diabetic properties of cashew plant parts were studied using differentiated C2C12 myoblasts (myotubes) and rat liver mitochondria. Hydroethanolic extract of cashew seed (CSE) and its active component, anacardic acid (AA), stimulated glucose transport into C2C12 myotubes in a concentration-dependent manner. Extracts of other parts (leaves, bark and apple) of cashew plant were inactive. Significant synergistic effect on glucose uptake with insulin was noticed at 100 μg/mL CSE. CSE and AA caused activation of adenosine monophosphate-activated protein kinase in C2C12 myotubes after 6 h of incubation. No significant effect was noticed on Akt and insulin receptor phosphorylation. Both CSE and AA exerted significant uncoupling of succinate-stimulated respiration in rat liver mitochondria. Activation of adenosine monophosphate-activated protein kinase by CSE and AA likely increases plasma membrane glucose transporters, resulting in elevated glucose uptake. In addition, the dysfunction of mitochondrial oxidative phosphorylation may enhance glycolysis and contribute to increased glucose uptake. These results collectively suggest that CSE may be a potential anti-diabetic nutraceutical. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Catalytic dephosphorylation of adenosine monophosphate (AMP) to form supramolecular nanofibers/hydrogels.

PubMed

Du, Xuewen; Li, Junfeng; Gao, Yuan; Kuang, Yi; Xu, Bing

2012-02-18

The use of enzyme to instruct the self-assembly of the nucleoside of adenosine in water provides a new class of molecular nanofibers/hydrogels as functional soft materials. This journal is © The Royal Society of Chemistry 2012

Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function.

PubMed

Bjursell, Magnus K; Blom, Henk J; Cayuela, Jordi Asin; Engvall, Martin L; Lesko, Nicole; Balasubramaniam, Shanti; Brandberg, Göran; Halldin, Maria; Falkenberg, Maria; Jakobs, Cornelis; Smith, Desiree; Struys, Eduard; von Döbeln, Ulrika; Gustafsson, Claes M; Lundeberg, Joakim; Wedell, Anna

2011-10-07

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

PubMed

Taylor, Raymond; Kotian, Pravin; Warren, Travis; Panchal, Rekha; Bavari, Sina; Julander, Justin; Dobo, Sylvia; Rose, Angela; El-Kattan, Yahya; Taubenheim, Brian; Babu, Yarlagadda; Sheridan, William P

2016-01-01

The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug's monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. All rights reserved.

Loss of ATM kinase activity leads to embryonic lethality in mice.

PubMed

Daniel, Jeremy A; Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V; You, Zhongsheng; Paull, Tanya T; Sleckman, Barry P; Feigenbaum, Lionel; Nussenzweig, André

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

Cyclic guanosine monophosphate does not inhibit gonadotropin-induced activation of mitogen-activated protein kinase 3/1 in pig cumulus-oocyte complexes.

PubMed

Blaha, Milan; Nemcova, Lucie; Prochazka, Radek

2015-01-07

Recent results indicate a key role for cyclic guanosine monophosphate (cGMP) in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles. The aim of our study was to determine whether the resumption of oocyte meiosis and expansion of cumulus cells in isolated pig cumulus-oocyte complexes (COCs) can be blocked by a high intracellular concentration of cGMP, and whether this effect is mediated by a cGMP-dependent inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1). The COCs were isolated from ovaries of slaughtered gilts and cultured in vitro in M199 supplemented with 5% fetal calf serum. The expression levels of the C-type natriuretic peptide (CNP) precursor (NPPC) and its receptor (NPR2) mRNAs during the culture of COCs were determined by real-time RT-PCR. To control the intracellular concentration of cGMP in the COCs, the culture medium was further supplemented with CNP or various concentrations of synthetic cGMP analogues; the concentration of cGMP in COCs was then assessed by ELISA. The effect of the drugs on oocyte maturation was assessed after 24 and 44 h of culture by determining nuclear maturation. The expansion of cumulus cells was assessed by light microscopy and the expression of cumulus expansion-related genes by real-time RT-PCR. A possible effect of cGMP on FSH-induced activation of MAPK3/1 was assessed by immunoblotting the COC proteins with phospho-specific and total anti-Erk1/2 antibodies. The COCs expressed NPPC and NPR2, the key components of cGMP synthesis, and produced a large amount of cGMP upon stimulation with exogenous CNP, which lead to a significant (P < 0.05) delay in oocyte meiotic resumption. The COCs also responded to cGMP analogues by inhibiting the resumption of oocyte meiosis. The inhibitory effect of cGMP on meiotic resumption was reversed by stimulating the COCs with FSH. However, high concentration of intracellular cGMP was not able to suppress FSH-induced activation of MAPK3/1 in cumulus cells, cumulus

Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

PubMed

Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

2016-11-15

The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. Copyright © 2016 Elsevier Ltd. All rights reserved.

Smoke Extract Impairs Adenosine Wound Healing. Implications of Smoke-Generated Reactive Oxygen Species

PubMed Central

Zimmerman, Matthew C.; Zhang, Hui; Castellanos, Glenda; O’Malley, Jennifer K.; Alvarez-Ramirez, Horacio; Kharbanda, Kusum; Sisson, Joseph H.; Wyatt, Todd A.

2013-01-01

Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 μM adenosine or the specific A2A receptor agonist, 5′-(N-cyclopropyl)–carboxamido–adenosine (CPCA; 10 μM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract–mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate–dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species–dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of

An evaluation of short-term corticosteroid response in perennial allergic rhinitis using histamine and adenosine monophosphate nasal challenge

PubMed Central

Wilson, Andrew M; Sims, Erika J; Orr, Linda C; Robb, Fiona; Lipworth, Brian J

2003-01-01

Aims To evaluate the role of AMP nasal challenge as a measure of short-term treatment response in patients receiving intranasal corticosteroids. Adenosine monophosphate (AMP) challenge has been shown to be a good inflammatory surrogate in the lower airways, but it has not been properly evaluated as a nasal challenge test. Methods Fourteen patients with perennial allergic rhinitis (PAR) were randomized to receive 2 weeks treatment with placebo (PL) or 200 µg intranasal mometasone furoate (MF) once daily in a randomized single-blind crossover study. AMP (25–800 mg ml−1) and histamine (0.25–8 mg ml−1) nasal challenge testing were performed after each treatment period with 30% decrease in minimal cross-sectional area (MCA). Domiciliary symptom data were collected. Results There was a significant (P < 0.05) improvement in PC30 MCA and nasal volume with AMP but not with histamine comparing MF vs PL. This amounted to a 2.8 (95% CI 1.5, 4.0) and 0.7 (95% CI −0.5, 1.9) doubling-dose change for AMP and histamine challenges, respectively. There were significant (P < 0.05) improvements in nasal symptoms and quality of life. Conclusions AMP nasal challenge using acoustic rhinometry may be a useful test to assess short-term treatment response in patient with PAR. PMID:12680883

Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan

Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-typemore » dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.« less

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

PubMed Central

Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

2013-01-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

Dehydroepiandrosterone activates AMP kinase and regulates GLUT4 and PGC-1α expression in C2C12 myotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokokawa, Takumi; Sato, Koji; Iwanaka, Nobumasa

Exercise and caloric restriction (CR) have been reported to have anti-ageing, anti-obesity, and health-promoting effects. Both interventions increase the level of dehydroepiandrosterone (DHEA) in muscle and blood, suggesting that DHEA might partially mediate these effects. In addition, it is thought that either 5′-adenosine monophosphate-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mediates the beneficial effects of exercise and CR. However, the effects of DHEA on AMPK activity and PGC-1α expression remain unclear. Therefore, we explored whether DHEA in myotubes acts as an activator of AMPK and increases PGC-1α. DHEA exposure increased glucose uptake but not the phosphorylation levelsmore » of Akt and PKCζ/λ in C2C12 myotubes. In contrast, the phosphorylation levels of AMPK were elevated by DHEA exposure. Finally, we found that DHEA induced the expression of the genes PGC-1α and GLUT4. Our current results might reveal a previously unrecognized physiological role of DHEA; the activation of AMPK and the induction of PGC-1α by DHEA might mediate its anti-obesity and health-promoting effects in living organisms. - Highlights: • We assessed whether dehydroepiandrosterone (DHEA) activates AMPK and PGC-1α. • DHEA exposure increased glucose uptake in C2C12 myotubes. • The phosphorylation levels of AMPK were elevated by DHEA exposure. • DHEA induced the expression of the genes PGC-1α and GLUT4. • AMPK might mediate the anti-obesity and health-promoting effects of DHEA.« less

Adenosine Kinase Modulates Root Gravitropism and Cap Morphogenesis in Arabidopsis1[W][OA

PubMed Central

Young, Li-Sen; Harrison, Benjamin R.; U.M., Narayana Murthy; Moffatt, Barbara A.; Gilroy, Simon; Masson, Patrick H.

2006-01-01

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-l-methionine pathway in the control of root gravitropism and cap morphogenesis. PMID:16891550

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase.

PubMed

Bello, Angelica M; Poduch, Ewa; Liu, Yan; Wei, Lianhu; Crandall, Ian; Wang, Xiaoyang; Dyanand, Christopher; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2008-02-14

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

Tangeretin triggers melanogenesis through the activation of melanogenic signaling proteins and sustained extracellular signal- regulated kinase in B16/F10 murine melanoma cells.

PubMed

Yoon, Hoon Seok; Ko, Hee-Chul; Kim, Sang Suk; Park, Kyung Jin; An, Hyun Joo; Choi, Young Hun; Kim, Se-Jae; Lee, Nam-Ho; Hyun, Chang-Gu

2015-03-01

In order to test the effectiveness of tangeretin at ameliorating melanoma and melanoma-associated depigmentation, western blotting was used to assess the melanin content of treated melanoma cells. Tangeretin, a 4',5,6,7,8-pentamethoxyflavone, was found to trigger intracellular melanin production in a concentration-dependent manner in B16/F10 murine melanoma cells. Melanin content increased 1.74-fold in response to treatment with 25 μM of tangeretin, compared to that in non-treated cells. Examination of melanogenic protein expression showed that tyrosinase, tyrosinase-related protein (TRP)-1, and extracellular signal-regulated kinase (ERK) 1/2 levels increased in a dose-dependent manner. Furthermore, the expression of cyclic adenosine monophosphate response element binding protein (CREB) and microphthalmia transcription factor (MITF) was increased by tangeretin in 1 h and 4 h, respectively. Tangeretin- upregulated melanogenesis was suppressed by ERK 1/2 inhibitor and not by ERK1 inhibitor. These results suggest that tangeretin has therapeutic potential for melanoma and melanoma-associated depigmentation because it can induce hyperpigmentation through the activation of melanogenic signaling proteins and initiation of sustained ERK2 expression.

Effects of cyclic adenosine monophosphate response element binding protein overexpression in the basolateral amygdala on behavioral models of depression and anxiety.

PubMed

Wallace, Tanya L; Stellitano, Kathryn E; Neve, Rachael L; Duman, Ronald S

2004-08-01

Chronic antidepressant administration increases the cyclic adenosine monophosphate response element binding protein (CREB) in the amygdala, a critical neural substrate involved in the physiologic responses to stress, fear, and anxiety. To determine the role of CREB in the amygdala in animal models of depression and anxiety, a viral gene transfer approach was used to selectively express CREB in this region of the rat brain. In the learned helplessness model of depression, induction of CREB in the basolateral amygdala after training decreased the number of escape failures, an antidepressant response. However, expression of CREB before training increased escape failures, and increased immobility in the forced swim test, depressive effects. Expression of CREB in the basolateral amygdala also increased behavioral measures of anxiety in both the open field test and the elevated plus maze, and enhanced cued fear conditioning. Taken together, these data demonstrate that CREB expression in the basolateral amygdala influences behavior in models of depression, anxiety, and fear. Moreover, in the basolateral amygdala, the temporal expression of CREB in relation to learned helplessness training, determines the qualitative outcome in this animal model of depression.

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue.

PubMed

Strouch, Marci B; Jackson, Edwin K; Mi, Zaichuan; Metes, Nicole A; Carey, Gale B

2005-06-01

Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling

PubMed Central

Nie, Jia; Liu, Xiaolei; Lilley, Brendan N.; Zhang, Hai; Pan, Y. Albert; Kimball, Scot R.; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S.; Sanes, Joshua R.; Han, Xiao; Shi, Yuguang

2013-01-01

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5′ adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic β-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5′-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling. PMID:23922392

SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling.

PubMed

Nie, Jia; Liu, Xiaolei; Lilley, Brendan N; Zhang, Hai; Pan, Y Albert; Kimball, Scot R; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S; Sanes, Joshua R; Han, Xiao; Shi, Yuguang

2013-08-20

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic β-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

Role of CNPase in the Oligodendrocytic Extracellular 2′,3′-cAMP-Adenosine Pathway

PubMed Central

Verrier, Jonathan D.; Jackson, Travis C.; Gillespie, Delbert G.; Janesko-Feldman, Keri; Bansal, Rashmi; Goebbels, Sandra; Nave, Klaus-Armin; Kochanek, Patrick M.; Jackson, Edwin K.

2014-01-01

Extracellular adenosine 3′,5′-cyclic monophosphate (3′,5′-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2′,3′-cAMP to adenosine. Here we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2′,3′-cAMP and their respective adenosine monophosphates (2′-AMP and 3′-AMP). Cells were also isolated from mice deficient in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2′,3′-cAMP to 2′-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3′-AMP was minimal in both oligodendrocytes and neurons. The production of 2′-AMP from 2′,3′-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2′-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3′,5′-cAMP-3′-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2′,3′-cAMP to 2′-AMP and inhibition of classic ecto-5′-nucleotidase (CD73) with α,β-methylene-adenosine-5′-diphosphate did not attenuate the conversion of 2′-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2′,3′-cAMP to 2-AMP in CNS cells. By reducing levels of 2′,3′-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant

(S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

PubMed Central

Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

2012-01-01

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH. PMID:22916194

(S)-α-chlorohydrin inhibits protein tyrosine phosphorylation through blocking cyclic AMP - protein kinase A pathway in spermatozoa.

PubMed

Zhang, Hao; Yu, Huan; Wang, Xia; Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

2012-01-01

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.

Phosphodiesterase 7 inhibitor reduced cognitive impairment and pathological hallmarks in a mouse model of Alzheimer's disease.

PubMed

Perez-Gonzalez, Rocio; Pascual, Consuelo; Antequera, Desiree; Bolos, Marta; Redondo, Miriam; Perez, Daniel I; Pérez-Grijalba, Virginia; Krzyzanowska, Agnieszka; Sarasa, Manuel; Gil, Carmen; Ferrer, Isidro; Martinez, Ana; Carro, Eva

2013-09-01

Elevated levels of amyloid beta (Aβ) peptide, hyperphosphorylation of tau protein, and inflammation are pathological hallmarks in Alzheimer's disease (AD). Phosphodiesterase 7 (PDE7) regulates the inflammatory response through the cyclic adenosine monophosphate signaling cascade, and thus plays a central role in AD. The aim of this study was to evaluate the efficacy of an inhibitor of PDE7, named S14, in a mouse model of AD. We report that APP/Ps1 mice treated daily for 4 weeks with S14 show: (1) significant attenuation in behavioral impairment; (2) decreased brain Aβ deposition; (3) enhanced astrocyte-mediated Aβ degradation; and (4) decreased tau phosphorylation. These effects are mediated via the cyclic adenosine monophosphate/cyclic adenosine monophosphate response element-binding protein signaling pathway, and inactivation of glycogen synthase kinase (GSK)3. Our data support the use of PDE7 inhibitors, and specifically S14, as effective therapeutic agents for the prevention and treatment of AD. Copyright © 2013 Elsevier Inc. All rights reserved.

N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors.

PubMed

Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32-35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR.

Adenosine receptor desensitization and trafficking.

PubMed

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2.

PubMed

Wang, Ping; Jiang, Yong; Wang, Yinsheng; Shyy, John Y; DeFea, Kathryn A

2010-09-21

Proteinase-activated-receptor-2 (PAR2) is a seven transmembrane receptor that can activate two separate signaling arms: one through Gαq and Ca2+ mobilization, and a second through recruitment of β-arrestin scaffolds. In some cases downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by β-arrestins, while in other cases the two pathways are synergistic; thus β-arrestins act as molecular switches capable of modifying the signal generated by the receptor. Here we demonstrate that PAR2 can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent Kinase Kinase β (CAMKKβ), while inhibiting AMPK through interaction with β-arrestins. The ultimate outcome of PAR2 activation depended on the cell type studied; in cultured fibroblasts with low endogenous β-arrestins, PAR2 activated AMPK; however, in primary fat and liver, PAR2 only activated AMPK in β-arrestin-2-/- mice. β-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKKβ under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR2 activation. Addition of recombinant β-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKKβ on Thr172. Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for β-arrestin in the inhibition of AMPK signaling, raising the possibility that β-arrestin-dependent PAR2 signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one.

Bronchodilator responses after methacholine and adenosine 5'-monophosphate (AMP) challenges in children with asthma: their relationships with eosinophil markers.

PubMed

Yoo, Young; Seo, Sung Chul; Kim, Young Il; Chung, Bo Hyun; Song, Dae Jin; Choung, Ji Tae

2012-09-01

Bronchodilator responsiveness (BDR) and eosinophilic inflammation are characteristic features of asthma. Objective. The aim of this study was to compare the relationships of BDR after methacholine challenge or adenosine 5'-monophosphate (AMP) challenge to blood eosinophil markers in children with asthma. Methacholine and AMP challenges were performed on 69 children with mild intermittent to moderate persistent asthma. BDR was calculated as the change in forced expiratory volume in 1 second, expressed as percentage change of the value immediately after the each challenge and the value after inhalation of salbutamol. Serum total IgE levels, blood eosinophil counts, and serum eosinophil cationic protein (ECP) levels were determined for each subject. A positive relationship between serum total IgE levels and BDR was found only after the AMP challenge (R(2) = 0.345, p = .001) rather than after the methacholine challenge (R(2) = 0.007, p = .495). Peripheral blood eosinophil counts correlated more significantly with BDR after AMP challenge (R(2) = 0.212, p = .001) than BDR after methacholine challenge (R(2) = 0.002, p = .724). Both BDR after methacholine challenge (R(2) = 0.063, p = .038) and BDR after AMP challenge (R(2) = 0.192, p = .001) were significantly correlated with serum ECP levels. BDR after AMP challenge may be more closely related to eosinophilic inflammation, compared with that after methacholine challenge.

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

PubMed

Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

2011-08-17

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

Relationships of methacholine and adenosine monophosphate responsiveness with serum vascular endothelial growth factor in children with asthma.

PubMed

Yoo, Young; Choi, Ic Sun; Byeon, Jung Hye; Lee, Seung Min; La, Kyong Suk; Choi, Byung Min; Park, Sang Hee; Choung, Ji Tae

2010-01-01

Airway hyperresponsiveness, which is a characteristic feature of asthma, is usually measured by means of bronchial challenge with direct or indirect stimuli. Vascular endothelial growth factor (VEGF) increases vascular permeability and angiogenesis, leads to mucosal edema, narrows the airway diameter, and reduces airway flow. To examine the relationships between serum VEGF level and airway responsiveness to methacholine and adenosine monophosphate (AMP) in children with asthma. Peripheral blood eosinophil counts, serum eosinophil cationic protein (ECP) concentrations, and serum VEGF concentrations were measured in 31 asthmatic children and 26 control subjects. Methacholine and AMP bronchial challenges were performed on children with asthma. Children with asthma had a significantly higher mean (SD) level of VEGF than controls (361.2 [212.0] vs 102.7 [50.0] pg/mL; P < .001). Blood eosinophil counts and serum ECP levels significantly correlated inversely with AMP provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC20) (r = -0.474, P =.01; r = -0.442, P =.03, respectively), but not with methacholine PC20 (r = -0.228, P = .26; r = -0.338, P =.10, respectively). Serum VEGF levels significantly correlated with airway responsiveness to AMP (r = -0.462; P = .009) but not to methacholine (r = -0.243; P = .19). Serum VEGF levels were increased in children with asthma and were related to airway responsiveness to AMP but not to methacholine. Increased VEGF levels in asthmatic children may result in increased airway responsiveness by mechanisms related to airway inflammation or increased permeability of airway vasculature.

Bronchial hyperresponsiveness to methacholine and adenosine monophosphate and the degree of atopy in children with allergic rhinitis.

PubMed

Kim, Chang Keun; Choi, Soo Jeon; Lee, Ju Kyung; Suh, Dong In; Koh, Young Yull

2011-01-01

nonasthmatic patients with allergic rhinitis often have bronchial hyperresponsiveness (BHR). Not only the presence but also the degree of atopy are important factors in BHR of patients with asthma. BHR is commonly evaluated by bronchial challenges using direct or indirect stimuli. to assess BHR to methacholine (direct) and to adenosine monophosphate (AMP) (indirect) in children with allergic rhinitis and to compare their relationships with the degree of atopy. methacholine and AMP challenges were performed in 88 children with allergic rhinitis, and a provocative concentration causing a 20% decrease in forced expiratory volume in 1 second (PC(20)) was calculated for each challenge. The degree of atopy was measured using serum total IgE levels, number of positive skin prick test results, and atopic scores (sum of graded wheal size). BHR to methacholine (PC(20) <8 mg/mL) and to AMP (PC(20) <200 mg/mL) was observed in 22 (25%) and 30 (34%) patients, respectively. No association was found between BHR to methacholine and any atopy parameter. In contrast, serum total IgE levels and atopic scores were higher in the group with BHR to AMP than in the group without BHR to AMP. Furthermore, a significant association was found between the degree of these 2 parameters and BHR to AMP (score for trend, P < .001 and P = .03, respectively). both BHR to methacholine and BHR to AMP were detected in a significant proportion of children with allergic rhinitis. The degree of atopy seems to be an important factor in BHR to AMP but not in BHR to methacholine.

The protection of meloxicam against chronic aluminium overload-induced liver injury in rats.

PubMed

Yang, Yang; He, Qin; Wang, Hong; Hu, Xinyue; Luo, Ying; Liang, Guojuan; Kuang, Shengnan; Mai, Shaoshan; Ma, Jie; Tian, Xiaoyan; Chen, Qi; Yang, Junqing

2017-04-04

The present study was designed to observe the protective effect and mechanisms of meloxicam on liver injury caused by chronic aluminium exposure in rats. The histopathology was detected by hematoxylin-eosin staining. The levels of prostaglandin E2, cyclic adenosine monophosphate and inflammatory cytokines were detected by enzyme linked immunosorbent assay. The expressions of cyclooxygenases-2, prostaglandin E2 receptors and protein kinase A were measured by western blotting and immunohistochemistry. Our experimental results showed that aluminium overload significantly damaged the liver. Aluminium also significantly increased the expressions of cyclooxygenases-2, prostaglandin E2, cyclic adenosine monophosphate, protein kinase A and the prostaglandin E2 receptors (EP1,2,4) and the levels of inflammation and oxidative stress, while significantly decreased the EP3 expression in liver. The administration of meloxicam significantly improved the impairment of liver. The contents of prostaglandin E2 and cyclic adenosine monophosphate were significantly decreased by administration of meloxicam. The administration of meloxicam also significantly decreased the expressions of cyclooxygenases-2 and protein kinase A and the levels of inflammation and oxidative stress, while significantly increased the EP1,2,3,4 expressions in rat liver. Our results suggested that the imbalance of cyclooxygenases-2 and downstream prostaglandin E2 signaling pathway is involved in the injury of chronic aluminium-overload rat liver. The protective mechanism of meloxicam on aluminium-overload liver injury is attributed to reconstruct the balance of cyclooxygenases-2 and downstream prostaglandin E2 signaling pathway.

[Qualitative analysis of bis-(3'-5')-cyclic dimeric adenosine monophosphate of Porphyromonas gingivalis by high performance liquid chromatography coupled with mass spectrometry].

PubMed

Yongmei, Tan; Xiaojun, Yang; Juan, Du; Wanghong, Zhao; Xiaodan, Chen; Jin, Hou

2016-06-01

To test whether Porphyromonas gingivalis (P. gingivalis) could produce bacterial signal molecule, bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and lay the foundation for explorations of its roles in life metabolism and periodontitis immunity of P. gingivalis. P. gingivalis standard strain ATCC33277 was used as the experimental strain to extract nucleic acids from the bacteria. Then, c-di-AMP was detected using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Subsequently, HPLC was used to validate the sample further. Based on the signal/noise (S/N) for 3 : 1, the limit of determination of HPLC-MS/MS for peak time of c-di-AMP standard substances was 7.49 min and nucleic acid extractions from P. gingivalis was 8.82 min (S/N > 3). Further confirmation of HPLC showed that nucleic acid extractions from both P. gingivalis and c-di-AMP standard substances pre- sented goal absorbent peaks at 15.7 min, with the same ultraviolet absorbent spectrum. The nucleic acid extrac- tions from P. gingivalis contained c-di-AMP, which shows that P. gingivalis could produce c-di-AMP.

DARPP chocolate: a caffeinated morsel of striatal signaling.

PubMed

Bastia, Elena; Schwarzschild, Michael A

2003-01-14

The psychomotor stimulant effects of caffeine, the most widely consumed psychoactive substance, are mediated through its antagonism of extracellular adenosine receptors in the basal ganglia. In the absence of caffeine, adenosine stimulates inhibitory striatopallidal neurons that suppress motor activity by binding to A2A receptors, thereby activating a cyclic adenosine 3',5'-monophosphate (cAMP) and protein kinase A signaling pathway. Bastia and Schwarzschild discuss recent research implicating DARRP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kilodaltons) as an attractive mediator of the sustained psychomotor stimulant effect seen with low doses of caffeine. They highlight the role of postsynaptic A2A receptor blockade, but leave open the possibility that antagonism of presynaptic or postsynaptic A1 receptors also contributes to DARPP-32-dependent psychomotor stimulation by caffeine.

Orotic aciduria and uridine monophosphate synthase: a reappraisal.

PubMed

Bailey, C J

2009-12-01

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation.

PubMed

Labesse, Gilles; Benkali, Khaled; Salard-Arnaud, Isabelle; Gilles, Anne-Marie; Munier-Lehmann, Hélène

2011-04-01

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

RNA Adenosine Deaminase ADAR1 Deficiency Leads to Increased Activation of Protein Kinase PKR and Reduced Vesicular Stomatitis Virus Growth Following Interferon Treatment

PubMed Central

Li, Zhiqun; Wolff, Karen C.; Samuel, Charles E.

2009-01-01

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells over expressing ADAR1 were utilized. Over expression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than ~10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was ~1 log10 further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment. PMID:19913273

Adenosine Monophosphate–Activated Protein Kinase Is Required for Pulmonary Artery Smooth Muscle Cell Survival and the Development of Hypoxic Pulmonary Hypertension

PubMed Central

Ibe, Joyce Christina F.; Zhou, Qiyuan; Chen, Tianji; Tang, Haiyang; Yuan, Jason X.-J.; Raj, J. Usha

2013-01-01

Human pulmonary artery smooth muscle cells (HPASMCs) express both adenosine monophosphate–activated protein kinase (AMPK) α1 and α2. We investigated the distinct roles of AMPK α1 and α2 in the survival of HPASMCs during hypoxia and hypoxia-induced pulmonary hypertension (PH). The exposure of HPASMCs to hypoxia (3% O2) increased AMPK activation and phosphorylation, and the inhibition of AMPK with Compound C during hypoxia decreased their viability and increased lactate dehydrogenase activity and apoptosis. Although the suppression of either AMPK α1 or α2 expression led to increased cell death, the suppression of AMPK α2 alone increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. It also resulted in the decreased expression of myeloid cell leukemia sequence 1 (MCL-1). The knockdown of MCL-1 or MCL-1 inhibitors increased caspase-3 activity and apoptosis in HPASMCs exposed to hypoxia. On the other hand, the suppression of AMPK α1 expression alone prevented hypoxia-mediated autophagy. The inhibition of autophagy induced cell death in HPASMCs. Our results suggest that AMPK α1 and AMPK α2 play differential roles in the survival of HPASMCs during hypoxia. The activation of AMPK α2 maintains the expression of MCL-1 and prevents apoptosis, whereas the activation of AMPK α1 stimulates autophagy, promoting HPASMC survival. Moreover, treatment with Compound C, which inhibits both isoforms of AMPK, prevented and partly reversed hypoxia-induced PH in mice. Taking these results together, our study suggests that AMPK plays a key role in the pathogenesis of pulmonary arterial hypertension, and AMPK may represent a novel therapeutic target for the treatment of pulmonary arterial hypertension. PMID:23668615

Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity

PubMed Central

Lovatt, Ditte; Xu, Qiwu; Liu, Wei; Takano, Takahiro; Smith, Nathan A.; Schnermann, Jurgen; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca2+ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity. PMID:22421436

Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma.

PubMed

Schuller, Hildegard M; Al-Wadei, Hussein A N; Majidi, Mourad

2008-10-01

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer in smokers and non-smokers that arises in most cases from small airway epithelial cells. PAC has a high mortality due to its aggressive behavior and resistance to cancer therapeutics. We have shown previously that the proliferation of human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D is stimulated by cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein and transactivation of the epidermal growth factor receptor and that this pathway is activated by beta-1-adrenoreceptors (beta(1)-ARs) and the non-genomic estrogen receptor beta. Our current in vitro studies with HPL1D and NCI-H322 cells showed that signaling via the gamma-amino butyric acid receptor (GABA(B)R) strongly inhibited base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory effects of gamma-amino butyric acid (GABA) were disinhibited by the GABA(B)R antagonist CGP-35348 or GABA(B)R knockdown. Immunohistochemical investigation of hamster lungs showed significant underexpression of GABA in animals with small airway-derived PACs induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These findings suggest that GABA may have tumor suppressor function in small airway epithelia and the PACs derived from them and that downregulation of GABA by NNK may contribute to the development of this cancer in smokers. Our findings suggest that marker-guided treatment with GABA or a GABA(B)R agonist of individuals with downregulated pulmonary GABA may provide a novel targeted approach for the prevention of PAC in smokers.

Guanosine-5'-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition.

PubMed

Molz, Simone; Dal-Cim, Tharine; Tasca, Carla I

2009-12-01

Guanine derivatives modulate the glutamatergic system through displacement of binding of glutamate to its receptors acting as antagonist of glutamate receptors in moderate to high micromolar concentrations. Guanosine-5'-monophosphate (GMP) is shown to be neuroprotective against glutamate- or oxygen/glucose deprivation-induced neurotoxicity and also against NMDA-induced apoptosis in hippocampal slices. However, in this study we are showing that high extracellular GMP concentrations (5mM) reduced cell viability in hippocampal brain slices. The toxic effect of GMP was not blocked by dipyridamole, a nucleoside transport inhibitor, nor mimicked by guanosine, suggesting an extracellular mode of action to GMP which does not involve its hydrolysis to guanosine. GMP-dependent cell damage was not blocked by P1 purinergic receptor antagonists, neither altered by adenosine A(1) or A(2A) receptor agonists. The blockage of the ionotropic glutamate receptors AMPA or NMDA, but not KA or metabotropic glutamate receptors, reversed the toxicity induced by GMP. GMP (5mM) induced a decrease in glutamate uptake into hippocampal slices, which was reversed by dl-TBOA. Therefore, GMP-induced hippocampal cell damage involves activation of ionotropic glutamate receptors and inhibition of glutamate transporters activity.

N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

PubMed Central

Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

2016-01-01

The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways.

PubMed

Lin, Y; Jia, R; Liu, Y; Gao, Y; Zeng, X; Kou, J; Yu, B

2014-12-01

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

Genetic Variation of the Kinases That Phosphorylate Tenofovir and Emtricitabine in Peripheral Blood Mononuclear Cells.

PubMed

Figueroa, Dominique B; Madeen, Erin P; Tillotson, Joseph; Richardson, Paul; Cottle, Leslie; McCauley, Marybeth; Landovitz, Raphael J; Andrade, Adriana; Hendrix, Craig W; Mayer, Kenneth H; Wilkin, Timothy; Gulick, Roy M; Bumpus, Namandjé N

2018-05-01

Tenofovir (TFV) disoproxil fumarate and emtricitabine (FTC) are used in combination for HIV treatment and pre-exposure prophylaxis (PrEP). TFV disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV. FTC and TFV are nucleoside/nucleotide reverse transcriptase inhibitors that upon phosphorylation to nucleotide triphosphate analogs competitively inhibit HIV reverse transcriptase. We previously demonstrated that adenylate kinase 2, pyruvate kinase, muscle and pyruvate kinase, liver and red blood cell phosphorylate TFV in peripheral blood mononuclear cells (PBMC). To identify the kinases that phosphorylate FTC in PBMC, siRNAs targeted toward kinases that phosphorylate compounds structurally similar to FTC were delivered to PBMC, followed by incubation with FTC and the application of a matrix-assisted laser desorption ionization-mass spectrometry method and ultra high performance liquid chromatography-UV to detect the formation of FTC phosphates. Knockdown of deoxycytidine kinase decreased the formation of FTC-monophosphate, while siRNA targeted toward thymidine kinase 1 decreased the abundance of FTC-diphosphate. Knockdown of either cytidine monophosphate kinase 1 or phosphoglycerate kinase 1 decreased the abundance of FTC-triphosphate. Next-generation sequencing of genomic DNA isolated from 498 HIV-uninfected participants in the HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 clinical study, revealed 17 previously unreported genetic variants of TFV or FTC phosphorylating kinases. Of note, four individuals were identified as simultaneous carriers of variants of both TFV and FTC activating kinases. These results identify the specific kinases that activate FTC in PBMC, while also providing further insight into the potential for genetic variation to impact TFV and FTC activation.

Determination of adenosine phosphates in rat gastrocnemius at various postmortem intervals using high performance liquid chromatography.

PubMed

Huang, Hong; Yan, Youyi; Zuo, Zhong; Yang, Lin; Li, Bin; Song, Yu; Liao, Linchuan

2010-09-01

Although the change in adenosine phosphate levels in muscles may contribute to the development of rigor mortis, the relationship between their levels and the onset and development of rigor mortis has not been well elucidated. In the current study, levels of the adenosine phosphates including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in gastrocnemius at various postmortem intervals of 180 rats from different death modes were detected by high performance liquid chromatography. The results showed that the levels of ATP and ADP significantly decreased along with the postmortem period of rats from different death mode whereas the AMP level remained the same. In addition, it was found that changes in the ATP levels in muscles after death correlated well with the development of rigor mortis. Therefore, the ATP level could serve as a reference parameter for the deduction of rigor mortis in forensic science.

Relaxation Effect of Abacavir on Rat Basilar Arteries

PubMed Central

Li, Rachel Wai Sum; Yang, Cui; Chan, Shun Wan; Hoi, Maggie Pui Man; Lee, Simon Ming Yuen; Kwan, Yiu Wa; Leung, George Pak Heng

2015-01-01

Background The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels. Methods The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5′ nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate. Results Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5’ nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries. Conclusion Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may

Removal of interfering nucleotides from brain extracts containing substance p. Effect of drugs on brain concentrations of substance p

PubMed Central

Laszlo, I.

1963-01-01

Several methods for removing interfering nucleotides, adenosine-5'-monophosphate and adenosine 5'-triphosphate from brain extracts have been studied. An enzymic method, using adenylic acid deaminase, has been found suitable. This deaminates adenosine monophosphate to 5'-inosinic acid, an inactive compound which does not influence the estimations of substance P. Owing to the adenosine triphosphatase content of the enzyme extract, adenosine triphosphate was also inactivated. For the estimation of adenosine monophosphate-deaminase activity, a simple colorimetric method is described which measures the ammonia liberated from adenosine monophosphate. Substance P in mouse brain extracts was estimated after treatment of the animals with various drugs, and after the enzymic removal of interfering nucleotides from the brain extracts. The drugs had no effect on the substance P content of mouse brain. The effect of drugs on the contractions of the guinea-pig ileum induced by substance P was also investigated, and the effect of drugs on the estimations of substance P in brain extracts is discussed. PMID:14066136

Chemical models and their mechanistic implications for the transformation of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase.

PubMed

Wu, Yuen-Jen; Liao, Chen-Chieh; Jen, Cheng-Hung; Shih, Yu-Chiao; Chien, Tun-Cheng

2010-07-14

The reactions of 6-cyano-1,3-dimethyluracil have been studied as chemical models to illustrate the mechanism for the transformation of 6-cyanouridine 5'-monophosphate (6-CN-UMP) to barbiturate ribonucleoside 5'-monophosphate (BMP) catalyzed by orotidine 5'-monophosphate decarboxylase (ODCase). The results suggest that the Asp residue in the ODCase active site plays the role of a general base in the transformation.

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

PubMed

Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

2016-05-01

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

PubMed

Abergel, Chantal; Blanc, Guillaume; Monchois, Vincent; Renesto, Patricia; Sigoillot, Cécile; Ogata, Hiroyuki; Raoult, Didier; Claverie, Jean-Michel

2006-11-01

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.

Discovery, synthesis, and structure-activity relationships of 20(S)-protopanaxadiol (PPD) derivatives as a novel class of AMPKα2β1γ1 activators.

PubMed

Liu, Junhua; Chen, Dakai; Liu, Peng; He, Mengna; Li, Jia; Li, Jingya; Hu, Lihong

2014-05-22

Adenosine 5'-monophosphate-activated protein kinase (AMPK) has been demonstrated as a promising drug target due to its regulatory function in glucose and lipid metabolism. 20(S)-protopanoxadiol (PPD) was firstly identified from high throughput screening as a small molecule activator of AMPK subtype α2β1γ1. In order to enhance its potency on AMPK, a series of PPD derivatives were synthesized and evaluated. Structure-activity relationship study showed that the amine derivatives at the 24-position (groups I-VI) can improve the potency (EC50: 0.7-2.3 μM) and efficacy (fold: 2.5-3.8). Among them, compounds 12 and 13 exhibited the best potency (EC50: 1.2 and 0.7 μM) and efficacy (fold: 3.7 and 3.8). Further study suggested the mechanism of AMPK activation may functioned at the allosteric position, resulting the inhibition of the lipid synthesis in HepG2 cell model. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: role of adenosine receptors.

PubMed

Maldonado, Claudio; Pushpakumar, Sathnur B; Perez-Abadia, Gustavo; Arumugam, Sengodagounder; Lane, Andrew N

2013-05-01

Ischemia-reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. The extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. PCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5'-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. Exogenous ATP administration to ischemic skeletal

A Genomic Response to Trace Fear Conditioning in the Amygdala of Female Rats After Developmental Exposure to Manganese

EPA Science Inventory

Increases in brain-derived neurotrophic factor (Bdnf), Ca2+/calmodulin-dependent protein kinase II alpha (Camk2a), and cyclic adenosine monophosphate (cAMP) response element binding (Creb1) gene expression have been associated with learning in a variety of different rodent studie...

Effect of curcumin (Curcuma longa extract) on LPS-induced acute lung injury is mediated by the activation of AMPK.

PubMed

Kim, Joungmin; Jeong, Seong-Wook; Quan, Hui; Jeong, Cheol-Won; Choi, Jeong-Il; Bae, Hong-Beom

2016-02-01

Curcumin, a biphenolic compound extracted from turmeric (Curcuma longa), possesses potent anti-inflammatory activity. The present study investigated whether curcumin could increase 5' adenosine monophosphate-activated protein kinase (AMPK) activity in macrophages and modulate the severity of lipopolysaccharide (LPS)-induced acute lung injury. Macrophages were treated with curcumin and then exposed (or not) to LPS. Acute lung injury was induced by intratracheal administration of LPS in BALB/c mice. Curcumin increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in a time- and concentration-dependent manner. Curcumin did not increase phosphorylation of liver kinase B1, a primary kinase upstream of AMPK. STO-609, an inhibitor of calcium(2+)/calmodulin-dependent protein kinase kinase, diminished curcumin-induced AMPK phosphorylation, but transforming growth factor-beta-activated kinase 1 inhibitor did not. Curcumin also diminished the LPS-induced increase in phosphorylation of inhibitory κB-alpha and the production of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein (MIP)-2, and interleukin (IL)-6 by macrophages. Systemic administration of curcumin significantly decreased the production of TNF-α, MIP-2, and IL-6 as well as neutrophil accumulation in bronchoalveolar lavage fluid, and also decreased pulmonary myeloperoxidase levels and the wet/dry weight ratio in mice subjected to LPS treatment. These results suggest that the protective effect of curcumin on LPS-induced acute lung injury is associated with AMPK activation.

Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A₂A receptor levels in rats subjected to neuroinflammation.

PubMed

Smith, Mark D; Bhatt, Dhaval P; Geiger, Jonathan D; Rosenberger, Thad A

2014-06-04

Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic

An easy and fast adenosine 5'-diphosphate quantification procedure based on hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry for determination of the in vitro adenosine 5'-triphosphatase activity of the human breast cancer resistance protein ABCG2.

PubMed

Wagmann, Lea; Maurer, Hans H; Meyer, Markus R

2017-10-27

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication. Copyright © 2017 Elsevier B.V. All rights reserved.

Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats.

PubMed

Rasmussen, Brittany A; Breen, Danna M; Luo, Ping; Cheung, Grace W C; Yang, Clair S; Sun, Biying; Kokorovic, Andrea; Rong, Weifang; Lam, Tony K T

2012-04-01

The duodenum senses nutrients to maintain energy and glucose homeostasis, but little is known about the signaling and neuronal mechanisms involved. We tested whether duodenal activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) is sufficient and necessary for cholecystokinin (CCK) signaling to trigger vagal afferent firing and regulate glucose production. In rats, we selectively activated duodenal PKA and evaluated changes in glucose kinetics during the pancreatic (basal insulin) pancreatic clamps and vagal afferent firing. The requirement of duodenal PKA signaling in glucose regulation was evaluated by inhibiting duodenal activation of PKA in the presence of infusion of the intraduodenal PKA agonist (Sp-cAMPS) or CCK1 receptor agonist (CCK-8). We also assessed the involvement of a neuronal network and the metabolic impact of duodenal PKA activation in rats placed on high-fat diets. Intraduodenal infusion of Sp-cAMPS activated duodenal PKA and lowered glucose production, in association with increased vagal afferent firing in control rats. The metabolic and neuronal effects of duodenal Sp-cAMPS were negated by coinfusion with either the PKA inhibitor H89 or Rp-CAMPS. The metabolic effect was also negated by coinfusion with tetracaine, molecular and pharmacologic inhibition of NR1-containing N-methyl-d-aspartate (NMDA) receptors within the dorsal vagal complex, or hepatic vagotomy in rats. Inhibition of duodenal PKA blocked the ability of duodenal CCK-8 to reduce glucose production in control rats, whereas duodenal Sp-cAMPS bypassed duodenal CCK resistance and activated duodenal PKA and lowered glucose production in rats on high-fat diets. We identified a neural glucoregulatory function of duodenal PKA signaling. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

AMPK activation by peri-sciatic nerve administration of ozone attenuates CCI-induced neuropathic pain in rats.

PubMed

Lu, Lijuan; Pan, Cailong; Chen, Lu; Hu, Liang; Wang, Chaoyu; Han, Yuan; Yang, Yanjing; Cheng, Zhixiang; Liu, Wen-Tao

2017-04-01

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. Ozone is widely used as an alternative therapy for many different pain conditions, with exact mechanisms still elusive. In this study, we found that a single peri-sciatic nerve injection of ozone decreased mechanical allodynia and thermal hyperalgesia, and normalized the phosphorylation of protein kinase C γ, N-methyl-D-aspartate receptor, and extracellular signal-regulated kinase in a chronic constriction injury (CCI) model in rat sciatic nerve. Meanwhile, ozone significantly suppressed CCI-induced activation of spinal microglia. More importantly, the anti-nociceptive effect of ozone depended on the activation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), which was proved by the fact that the phosphorylated AMPK level increased during the ozone therapy and AMPK antagonist abolished the effect of ozone in vivo and in vitro. In addition, direct injection of AMPK agonist could replicate the anti-nociceptive effect of ozone in CCI rats. In conclusion, our observations indicate that peri-sciatic nerve injection of ozone activates AMPK to attenuate CCI-induced neuropathic pain. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity.

PubMed

Poppe, Daniel; Doerr, Jonas; Schneider, Marion; Wilkens, Ruven; Steinbeck, Julius A; Ladewig, Julia; Tam, Allison; Paschon, David E; Gregory, Philip D; Reik, Andreas; Müller, Christa E; Koch, Philipp; Brüstle, Oliver

2018-06-01

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477-486. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

PubMed Central

Pani, Amar K.; Jiao, Yun; Sample, Kenneth J.; Smeyne, Richard J.

2014-01-01

Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models. PMID:24642754

Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

PubMed Central

Wall, Mark J; Dale, Nicholas

2013-01-01

The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

PubMed

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Caffeine and contraction synergistically stimulate 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle

PubMed Central

Tsuda, Satoshi; Egawa, Tatsuro; Kitani, Kazuto; Oshima, Rieko; Ma, Xiao; Hayashi, Tatsuya

2015-01-01

5′-Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a key mediator of contraction-stimulated insulin-independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr172 phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser473 phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction-stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin-independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction. PMID:26471759

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

PubMed

Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

1997-04-15

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other

Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major.

PubMed

Hossain, Md Sakhawat; Koshio, Shunsuke; Ishikawa, Manabu; Yokoyama, Saichiro; Sony, Nadia Mahjabin

2016-09-01

Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream. A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4 g) for 56 days. The results indicated that dietary AMP supplements tended to improve growth performances. One of the best ones was found in diet group AMP-0.2, followed by diet groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter, protein and lipid) also improved by AMP supplementation and the significantly highest dry matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P < 0.05) greater in fish fed diet groups AMP-0.4 and AMP-0.8. Total serum protein, lysozyme activity and agglutination antibody titer were also increased (P > 0.05) by dietary supplementation. In contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP supplemented diets had better improvement (P < 0.05) in body lipid contents, condition factor, hematocrit content and glutamyl oxaloacetic transaminase (GOT) level than the control group. Supplementation also improved both freshwater and oxidative stress resistances. Interestingly, the fish fed diet groups AMP-0.2 and AMP-0.4 showed the least oxidative stress condition. Finally it is concluded that, dietary AMP supplementation enhanced the growth, digestibility, immune response and stress resistance of red sea bream. The regression analysis revealed that a dietary AMP supplementation between 0.2 and 0.4% supported weight gain and

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

PubMed

Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

2016-12-30

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus*

PubMed Central

Bowman, Lisa; Zeden, Merve S.; Kaever, Volkhard

2016-01-01

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5′-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. PMID:27834680

Crystal Structure of Human Nicotinamide Riboside Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan,J.; Xiang, S.; Tong, L.

2007-01-01

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheetmore » core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.« less

Crystal structure of human nicotinamide riboside kinase.

PubMed

Khan, Javed A; Xiang, Song; Tong, Liang

2007-08-01

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A*

PubMed Central

Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P.

2015-01-01

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min−1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

PubMed Central

Guerrero-Mendiola, Carlos; García-Trejo, José J.; Encalada, Rusely; Saavedra, Emma

2017-01-01

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. PMID:28686591

Adenosine A2A receptor agonists with potent antiplatelet activity.

PubMed

Fuentes, Eduardo; Fuentes, Manuel; Caballero, Julio; Palomo, Iván; Hinz, Sonja; El-Tayeb, Ali; Müller, Christa E

2018-05-01

Selected adenosine A 2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC 50 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC 50 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC 50 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC 50 0.97±0.07 µmol/L) and equipotent to NECA (EC 50 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K i values determined in radioligand-binding studies were not predictive of the A 2A agonists' antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A 2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A 2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A 2A receptor agonists.

Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression.

PubMed

Liang, Xiaonan; Yong, Zheng; Su, Ruibin

2018-06-11

Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression. Copyright © 2018 Elsevier B.V. All rights reserved.

Adenosine and preeclampsia.

PubMed

Salsoso, Rocío; Farías, Marcelo; Gutiérrez, Jaime; Pardo, Fabián; Chiarello, Delia I; Toledo, Fernando; Leiva, Andrea; Mate, Alfonso; Vázquez, Carmen M; Sobrevia, Luis

2017-06-01

Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

PubMed Central

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-01-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve αCD3/CD28-stimulated CD8 cells. Consequently, αCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs. PMID:19740334

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

PubMed

Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

2009-09-01

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

Parallel effect of 4-octylphenol and cyclic adenosine monophosphate (cAMP) alters steroidogenesis, cell viability and ROS production in mice Leydig cells.

PubMed

Jambor, Tomas; Greifova, Hana; Kovacik, Anton; Kovacikova, Eva; Tvrda, Eva; Forgacs, Zsolt; Massanyi, Peter; Lukac, Norbert

2018-05-01

Over the last decade, there is growing incidence of male reproductive malfunctions. It has been documented that numerous environmental contaminants, such as endocrine disruptors (EDs) may adversely affect the reproductive functions of humans as well as wildlife species. The aim of this in vitro study was to examine the effects of 4-octylphenol (4-OP) on the steroidogenesis in mice Leydig cells. We evaluated the impact of this endocrine disruptor on the cholesterol levels and hormone secretion in a primary culture. Subsequently, we determined the cell viability and generation of reactive oxygen species (ROS) following 4-OP treatment. Isolated mice Leydig cells were cultured in the presence of different 4-OP concentrations (0.04-5.0 μg/mL) and 1 mM cyclic adenosine-monophosphate during 44 h. Cholesterol levels were determined from the culture medium using photometry. Quantification of steroid secretion was performed by enzyme-linked immunosorbent assay. The cell viability was assessed using the metabolic activity assay, while ROS production was assessed by the chemiluminescence technique. Slightly increased cholesterol levels were recorded following exposure to the whole applied range of 4-OP, without significant changes (P>0.05). In contrast, the secretion of steroid hormones, specifically dehydroepiandrosterone, androstenedione, and testosterone was decreased following exposure to 4-OP. Experimental doses of 4-OP did not affect cell viability significantly; however a moderate decrease was recorded following the higher doses (2.5 and 5.0 μg/mL) of 4-OP. Furthermore, relative treatment of 4-OP (5.0 μg/mL) caused a significant (P < 0.001) ROS overproduction in the exposed cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adenosine monophosphate is elevated in the bronchoalveolar lavage fluid of mice with acute respiratory toxicity induced by nanoparticles with high surface hydrophobicity.

PubMed

Dailey, Lea Ann; Hernández-Prieto, Raquel; Casas-Ferreira, Ana Maria; Jones, Marie-Christine; Riffo-Vasquez, Yanira; Rodríguez-Gonzalo, Encarnación; Spina, Domenico; Jones, Stuart A; Smith, Norman W; Forbes, Ben; Page, Clive; Legido-Quigley, Cristina

2015-02-01

Inhaled nanomaterials present a challenge to traditional methods and understanding of respiratory toxicology. In this study, a non-targeted metabolomics approach was used to investigate relationships between nanoparticle hydrophobicity, inflammatory outcomes and the metabolic fingerprint in bronchoalveolar fluid. Measures of acute lung toxicity were assessed following single-dose intratracheal administration of nanoparticles with varying surface hydrophobicity (i.e. pegylated lipid nanocapsules, polyvinyl acetate nanoparticles and polystyrene beads; listed in order of increasing hydrophobicity). Broncho-alveolar lavage (BAL) fluid was collected from mice exposed to nanoparticles at a surface area dose of 220 cm(2) and metabolite fingerprints were acquired via ultra pressure liquid chromatography-mass spectrometry-based metabolomics. Particles with high surface hydrophobicity were pro-inflammatory. Multivariate analysis of the resultant small molecule fingerprints revealed clear discrimination between the vehicle control and polystyrene beads (p < 0.05), as well as between nanoparticles of different surface hydrophobicity (p < 0.0001). Further investigation of the metabolic fingerprints revealed that adenosine monophosphate (AMP) concentration in BAL correlated with neutrophilia (p < 0.01), CXCL1 levels (p < 0.05) and nanoparticle surface hydrophobicity (p < 0.001). Our results suggest that extracellular AMP is an intermediary metabolite involved in adenine nucleotide-regulated neutrophilic inflammation as well as tissue damage, and could potentially be used to monitor nanoparticle-induced responses in the lung following pulmonary administration.

Single and short-term dosing effects of levocetirizine on adenosine monophosphate bronchoprovocation in atopic asthma

PubMed Central

Lee, Daniel K C; Gray, Robert D; Wilson, Andrew M; Robb, Fiona M; Soutar, Patricia C; Lipworth, Brian J

2004-01-01

Aims Adenosine monophosphate (AMP) acts indirectly via primed airway mast cells to induce bronchial hyper-responsiveness, which in turn correlates with eosinophilic asthmatic inflammation and atopic disease expression. We evaluated single and short-term dosing effects of a modern histamine H1-receptor antagonist, levocetirizine, given at the usual clinically recommended dose, on the primary outcome of AMP bronchoprovocation. Methods Fifteen atopic asthmatics were randomized in double-blind, cross-over fashion to receive for 1 week either levocetirizine 5 mg or placebo. There was a 1-week washout period prior to each randomized treatment. The provocative concentration of AMP producing a 20% fall in FEV1 (PC20) was measured after each washout at baseline and at 4–6 h following the first and last doses of each randomized treatment. Results Baseline mean ± SEM values after washout prior to each randomized treatment comparing levocetirizine vs placebo were not significantly different for prechallenge FEV1 (% predicted) 83 ± 4 vs 82 ± 4, or AMP PC20 (mg ml−1) 45 ± 24 vs 45 ± 22, respectively. Airway calibre as prechallenge FEV1 for levocetirizine vs placebo was not significantly different following the first dose 86 ± 4 vs 82 ± 4, or the last dose 85 ± 4 vs 83 ± 4, respectively. There were significant improvements (P< 0.05) in AMP PC20 comparing levocetirizine vs placebo following the first dose 123 ± 73 vs 48 ± 24, a 1.4 doubling dilution difference (95% CI 0.8, 1.9), and the last dose 127 ± 74 vs 53 ± 29, a 1.2 doubling dilution difference (95% CI 0.5, 2.0). AMP PC20 was also improved (P< 0.05) by the first and last doses of levocetirizine but not placebo, vs respective baseline values, with there being no difference in the degree of protection between first and last doses. Conclusions Single and short-term dosing with levocetirizine conferred similar improvements in bronchial hyper-responsiveness to AMP challenge, which was unrelated to prechallenge

Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

PubMed Central

Raju, Sammeta V.; Wang, Guoshun

2012-01-01

Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (ISC) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A2B adenosine receptor (A2BAR), largely abolished the adenosine-stimulated chloride transport, suggesting that A2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections. PMID:22442662

Colorimetric detection of mercury(II) in a high-salinity solution using gold nanoparticles capped with 3-mercaptopropionate acid and adenosine monophosphate.

PubMed

Yu, Cheng-Ju; Tseng, Wei-Lung

2008-11-04

A new colorimetric sensor for sensing Hg2+ in a high-salinity solution has been developed using gold nanoparticles (AuNPs) decorated with 3-mercaptopropionate acid (MPA) and adenosine monophosphate (AMP). Because of the high negative charge density of AMP on each AuNP surface, MPA/AMP-capped AuNPs are well dispersed in a high-salt solution. In contrast, the aggregation of MPA-capped AuNPs was induced by sodium ions, which shield the negative charges of the carboxylic groups of MPA. Through the coordination between the carboxylic group of MPA and Hg2+, the selectivity of MPA/AMP-capped AuNPs for Hg2+ in a high-salt solution is remarkably high over that of the other metals without the addition of a masking agent or a change in the temperature. We have carefully investigated the effect of the AMP concentration on the stability and sensitivity of MPA/AMP-capped AuNPs. Under optimum conditions, the lowest detectable concentration of Hg2+ using this probe was 500 nM on the basis of the measurement of the ratio of absorption at 620 nm to that at 520 nm. The sensitivity to Hg2+ can be further improved by modifying the MPA/AMP-capped AuNPs with highly fluorescent rhodamine 6G (R6G). By monitoring the fluorescence enhancement, the lowest detectable concentration of Hg2+ using R6G/MPA/AMP-capped AuNPs was 50 nM.

Methacholine and adenosine 5'-monophosphate (AMP) responsiveness, and the presence and degree of atopy in children with asthma.

PubMed

Suh, Dong I; Lee, Ju K; Kim, Chang K; Koh, Young Y

2011-02-01

The relationship between atopy and bronchial hyperresponsiveness (BHR), both key features of asthma, remains to be clarified. BHR is commonly evaluated by bronchial challenges using direct and indirect stimuli. The aim of this study was to investigate the degree of BHR to methacholine (direct stimulus) and adenosine 5'-monophosphate (AMP) (indirect stimulus) according to the presence and degree of atopy in children with asthma. We performed a retrospective analysis of data from 120 children presenting with a diagnosis of asthma. These children were characterized by skin-prick tests (SPTs), spirometry and bronchial challenges with methacholine and AMP. Atopy was defined by at least one positive reaction to SPTs, and its degree was measured using serum total IgE levels, number of positive SPTs and atopic scores (sum of graded wheal size). A provocative concentration causing a 20% decline in FEV(1) (PC(20) ) was determined for each challenge. Patients with atopy(n=94) had a significantly lower AMP PC(20) than non-atopic patients (n=26), whereas methacholine PC(20) was not different between the two groups. Among the patients with atopy, there was no association between methacholine PC(20) and any atopy parameter. In contrast, a significant association was found between AMP PC(20) and the degree of atopy reflected in serum total IgE, number of positive SPTs and atopic scores (anova trend test, p=0.002, 0.001, 0.003, respectively). AMP responsiveness was associated with the presence and degree of atopy, whereas such a relationship was not observed for methacholine responsiveness. These findings suggest that atopic status may be better reflected by bronchial responsiveness assessed by AMP than by methacholine. © 2011 John Wiley & Sons A/S.

Adenosine monophosphate is not superior to histamine for bronchial provocation test for assessment of asthma control and symptoms.

PubMed

Wu, Fan; Guan, Wei-Jie; Gao, Yi; An, Jia-Ying; Xie, Yan-Qing; Liu, Wen-Ting; Yu, Xin-Xin; Zheng, Jin-Ping

2017-07-01

Adenosine monophosphate (AMP) may reflect airway inflammation and hyperresponsiveness, but relationship between AMP and histamine (His, a conventional stimulus) bronchial provocation test (BPT) in asthma is not fully elucidated. To compare both BPTs and determine their utility in reflecting changes of asthmatic symptoms. BPTs were performed in a cross-over fashion, at 2-4 day intervals. Cumulative doses eliciting 20% FEV 1 fall (PD 20 FEV 1 ), diagnostic performance and adverse events (AEs) were compared. Patients with PD 20 FEV 1 lower than geometric mean were defined as responders, otherwise poor responders. Patients with uncontrolled and partly controlled asthma, who maintained their original inhaled corticosteroids therapy, underwent reassessment of airway responsiveness and asthmatic symptoms 3 and 6 months after. Nineteen uncontrolled, 22 partly controlled and 19 controlled asthmatic patients and 24 healthy subjects were recruited. Lower PD 20 FEV 1 geometric means were associated with poorer asthma control in His-BPT (0.424 μmol vs 1.684 μmol vs 3.757 μmol), but not AMP-BPT (11.810 μmol vs 7.781 μmol vs 10.220 μmol). Both BPTs yielded similar overall diagnostic performance in asthma (area under curve: 0.842 in AMP-BPT vs 0.850 in His-BPT). AEs, including wheezing and tachypnea, were similar and mild. Ten patients with uncontrolled and 10 partly controlled asthma were followed-up. At months 3 and 6, we documented an increase in PD 20 FEV 1 -AMP and PD 20 FEV 1 -His, which did not correlate with reduction asthmatic symptom scores. This overall applied in responders and poor responders of AMP-BPT and His-BPT. Despite higher screening capacity of well-controlled asthma, AMP-BPT confers similar diagnostic performance and safety with His-BPT. AMP-BPT might not preferentially reflect changes asthmatic symptoms. © 2015 John Wiley & Sons Ltd.

The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

PubMed Central

Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

2014-01-01

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

PubMed

Van de Werve, G; Hers, H G

1979-01-15

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

3',5'-cIMP as Potential Second Messenger in the Vascular Wall.

PubMed

Leung, Susan W S; Gao, Yuansheng; Vanhoutte, Paul M

2017-01-01

Traditionally, only the 3',5'-cyclic monophosphates of adenosine and guanosine (produced by adenylyl cyclase and guanylyl cyclase, respectively) are regarded as true "second messengers" in the vascular wall, despite the presence of other cyclic nucleotides in different tissues. Among these noncanonical cyclic nucleotides, inosine 3',5'-cyclic monophosphate (cIMP) is synthesized by soluble guanylyl cyclase in porcine coronary arteries in response to hypoxia, when the enzyme is activated by endothelium-derived nitric oxide. Its production is associated with augmentation of vascular contraction mediated by stimulation of Rho kinase. Based on these findings, cIMP appears to meet most, if not all, of the criteria required for it to be accepted as a "second messenger," at least in the vascular wall.

Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

PubMed Central

Rattan, R; Giri, S; Hartmann, LC; Shridhar, V

2011-01-01

Abstract Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways. PMID:19874425

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Conformational Changes in Orotidine 5-Monophosphate Decarboxylase: "Remote" Residues That Stabilize the Active Conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wood, B.; Amyes, T; Fedorov, A

2010-01-01

The structural factors responsible for the extraordinary rate enhancement ({approx}10{sup 17}) of the reaction catalyzed by orotidine 5{prime}-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and 'clamps' the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a 'remote' structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases k{sub cat}/K{sub m} with a minimal effect on k{sub cat},more » providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5{prime}-phosphate group of orotidine 5{prime}-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion.« less

Role of adenosine transport in gestational diabetes-induced l-arginine transport and nitric oxide synthesis in human umbilical vein endothelium

PubMed Central

Vásquez, Gustavo; Sanhueza, Felipe; Vásquez, Rodrigo; González, Marcelo; Martín, Rody San; Casanello, Paola; Sobrevia, Luis

2004-01-01

Gestational diabetes is associated with increased l-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial l-arginine/NO pathway via A2 purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 μm), and intracellular l-arginine (1.9 mm) and l-citrulline (0.7 mm) levels compared with normal cells (0.05 μm, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 μm nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44mapk activation, and were blocked by the A2a purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the l-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A2a purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of l-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus. PMID:15272035

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

PubMed

Krungkrai, J; Wutipraditkul, N; Prapunwattana, P; Krungkrai, S R; Rochanakij, S

2001-12-15

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes. (c)2001 Elsevier Science.

AMP-guided tumour-specific nanoparticle delivery via adenosine A1 receptor.

PubMed

Dai, Tongcheng; Li, Na; Han, Fajun; Zhang, Hua; Zhang, Yuanxing; Liu, Qin

2016-03-01

Active targeting-ligands have been increasingly used to functionalize nanoparticles for tumour-specific clinical applications. Here we utilize nucleotide adenosine 5'-monophosphate (AMP) as a novel ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for tumour-targeted imaging. We demonstrate that AMP-conjugated NPs (NPs-AMP) efficiently bind to and are following internalized into colon cancer cell CW-2 and breast cancer cell MDA-MB-468 in vitro. RNA interference and inhibitor assays reveal that the targeting effects mainly rely on the specific binding of AMP to adenosine A1 receptor (A1R), which is greatly up-regulated in cancer cells than in matched normal cells. More importantly, NPs-AMP specifically accumulate in the tumour site of colon and breast tumour xenografts and are further internalized into the tumour cells in vivo via tail vein injection, confirming that the high in vitro specificity of AMP can be successfully translated into the in vivo efficacy. Furthermore, NPs-AMP exhibit an active tumour-targeting behaviour in various colon and breast cancer cells, which is positively related to the up-regulation level of A1R in cancer cells, suggesting that AMP potentially suits for more extensive A1R-overexpressing cancer models. This work establishes AMP to be a novel tumour-targeting ligand and provides a promising strategy for future diagnostic or therapeutic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Uridine monophosphate kinase as potential target for tuberculosis: from target to lead identification.

PubMed

Arvind, Akanksha; Jain, Vaibhav; Saravanan, Parameswaran; Mohan, C Gopi

2013-12-01

Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis (TB) disease, which has affected approximately 2 billion people worldwide. Due to the emergence of resistance towards the existing drugs, discovery of new anti-TB drugs is an important global healthcare challenge. To address this problem, there is an urgent need to identify new drug targets in Mtb. In the present study, the subtractive genomics approach has been employed for the identification of new drug targets against TB. Screening the Mtb proteome using the Database of Essential Genes (DEG) and human proteome resulted in the identification of 60 key proteins which have no eukaryotic counterparts. Critical analysis of these proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database revealed uridine monophosphate kinase (UMPK) enzyme as a potential drug target for developing novel anti-TB drugs. Homology model of Mtb-UMPK was constructed for the first time on the basis of the crystal structure of E. coli-UMPK, in order to understand its structure-function relationships, and which would in turn facilitate to perform structure-based inhibitor design. Furthermore, the structural similarity search was carried out using physiological inhibitor UTP of Mtb-UMPK to virtually screen ZINC database. Retrieved hits were further screened by implementing several filters like ADME and toxicity followed by molecular docking. Finally, on the basis of the Glide docking score and the mode of binding, 6 putative leads were identified as inhibitors of this enzyme which can potentially emerge as future drugs for the treatment of TB.

Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction.

PubMed

Ozawa, Eijiro

2011-01-01

It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was how to connect these two currents. We found that low concentrations of Ca ions (10(-7)-10(-4) M) released from the sarcoplasmic reticulum for the regulation of muscle contraction simultaneously reversibly activate phosphorylase kinase, the enzyme regulating glycogenolysis. Moreover, we found that adenosine 3',5'-monophosphate (cyclic AMP), which is already known to activate muscle phosphorylase kinase, is not effective in the absence of such concentrations of Ca ions. Thus, cyclic AMP is not effective by itself alone and only modifies the activation process in the presence of Ca ions (at that time, cyclic AMP-dependent protein kinase had not yet been identified). After a while, it turned out that our works have not only provided the solution to the above problem on muscle physiology, but have also been considered as the first report of Ca-dependent protein phosphorylation, which is one of the central problems in current cell biology. Phosphorylase kinase is the first protein kinase to phosphorylate a protein resulting in the change in the function of the phosphorylated protein, as shown by Krebs and Fischer. Our works further showed that this protein kinase is regulated in a Ca-dependent manner. Accordingly, our works introduced the concept of low concentrations of Ca ions, which were first identified as the regulatory substance of muscle contraction, to the vast field of Ca biology including signal transduction.

Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

PubMed Central

Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

2013-01-01

The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylation may be elevated during sleep. Here, we report that cAMP, phospho-p44/42 MAPK and phospho-CREB are higher in rapid eye movement (REM) sleep compared to awake mice but are not elevated in non-rapid eye movement (NREM) sleep. This peak of activity during REM sleep does not occur in mice lacking calmodulin-stimulated adenylyl cyclases, a mouse strain that learns but cannot consolidate hippocampus-dependent memory. We conclude that a preferential increase in cAMP, MAPK activity and CREB phosphorylation during REM sleep may contribute to hippocampus-dependent memory consolidation. PMID:23575844

syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction

PubMed Central

El-Hillal, O.; Kurosaki, T.; Yamamura, H.; Kinet, J.-P.; Scharenberg, A. M.

1997-01-01

Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well. PMID:9050880

[Role of cyclic adenosine monophosphate(cAMP) in the regulation of intestinal epithelial barrier function under hypoxia].

PubMed

Yang, Yang; Wang, Wen-Sheng; Qiu, Yuan; Sun, Li-Hua; Yang, Hua

2013-05-01

To investigate the role of cyclic adenosine monophosphate(cAMP) in the regulation of intestinal epithelial barrier function under hypoxia. Intestinal epithelial barrier was established by Caco-2 monolayers. Cells were divided into four groups: normoxia (Nx), normoxia plus Forskolin(Nx+FSK), hypoxia(Hx), hypoxia plus SQ22536(Hx+SQ22536). cAMP concentrations of different groups were assessed by cAMP enzyme immunoassay kit. RT-PCR and Western blotting were used to detect the mRNA and protein expressions of claudin-1 and occludin under normoxic and hypoxic condition. Caco-2 monolayers were grown on Millicell filters, and transepithelial electrical resistance(TER) was measured using a Millipore electric resistance system. The concentration of cAMP under hypoxic conditions(Hx group) was higher compared with Nx group [(6.30±0.50) pmol/L vs. (2.38±0.18) pmol/L, P<0.01]. At the same time, both mRNA and protein expressions of claudin-1 and occluding were lower in Hx group than those in Nx group(all P<0.05). TER decreased by 76.30±0.64(P<0.01). When the monolayers were exposed to hypoxia plus SQ22536 (Hx+SQ22536 group), the concentration of cAMP was(2.12±0.23) pmol/L, which was lower than that under hypoxic conditions(Hx group, P<0.01). Both mRNA and protein expressions of claudin-1 and occludin were higher compared to Hx group (all P<0.01). TER increased by 32.96±2.16 (P<0.05). When Caco-2 cells are exposed to hypoxia, barrier function, claudin-1 and occludin expression are diminished in parallel with a high level of intracellular cAMP compared with the normoxic condition. Inhibition of the intracellular cAMP level under hypoxia can maintain the intestinal epithelial function through regulating the claudin-1 and occludin expression and attenuate the permeability of intestinal mucosa.

Reconsideration of the sequence of rigor mortis through postmortem changes in adenosine nucleotides and lactic acid in different rat muscles.

PubMed

Kobayashi, M; Takatori, T; Iwadate, K; Nakajima, M

1996-10-25

We examined the changes in adenosine triphosphate (ATP), lactic acid, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in five different rat muscles after death. Rigor mortis has been thought to occur simultaneously in dead muscles and hence to start in small muscles sooner than in large muscles. In this study we found that the rate of decrease in ATP was significantly different in each muscle. The greatest drop in ATP was observed in the masseter muscle. These findings contradict the conventional theory of rigor mortis. Similarly, the rates of change in ADP and lactic acid, which are thought to be related to the consumption or production of ATP, were different in each muscle. However, the rate of change of AMP was the same in each muscle.

UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy.

PubMed

Wang, Li; Burmeister, Brian T; Johnson, Keven R; Baillie, George S; Karginov, Andrei V; Skidgel, Randal A; O'Bryan, John P; Carnegie, Graeme K

2015-05-01

Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic β-adrenergic receptor (β-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3',5'-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic β-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity. Published by Elsevier Inc.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

PubMed

Frank, K B; Cheng, Y C

1986-02-05

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

Modulation of bicarbonate secretion in rabbit duodenum: the role of calcium.

PubMed

Hogan, D L; Yao, B; Isenberg, J I

1998-01-01

Surface epithelial bicarbonate secretion protects the proximal duodenum from acid peptic injury. Cyclic adenosine monophosphate and calcium serve as intracellular mediators of intestinal transport. Experiments were performed to examine whether calcium participates in duodenal bicarbonate transport. Stripped duodenal mucosa from rabbits was studied in Ussing chambers. HCO3- transport was stimulated by the calcium ionophore A23187, carbachol, vasoactive intestinal peptide, prostaglandin E2, dibutyryl-cyclic adenosine monophosphate, and electrical field stimulation. A23187 stimulated HCO3- secretion and Isc; tetrodotoxin failed to inhibit this effect. The calcium-channel blocker verapamil abolished HCO3- secretion stimulated by carbachol, vasoactive intestinal peptide, and electrical field stimulation, but failed to alter basal, prostaglandin E2- or dibutyryl-cyclic adenosine monophosphate-stimulated HCO3- secretion. Therefore, calcium is likely required during stimulation of duodenal epithelial HCO3- transport by carbachol, vasoactive intestinal peptide, and electrical field stimulation. Prostaglandin E2 and dibutyryl-cyclic adenosine monophosphate appear to activate duodenal HCO3- secretion by a calcium-independent pathway(s).

Mechanisms of Cachexia in Chronic Disease States.

PubMed

Yoshida, Tadashi; Delafontaine, Patrice

2015-10-01

Sarcopenia and cachexia are muscle wasting syndromes associated with aging and with many chronic diseases, such as congestive heart failure (CHF), diabetes, cancer, chronic obstructive pulmonary disease and chronic kidney disease (CKD). While mechanisms are complex, these conditions are often accompanied by elevated angiotensin II (Ang II). Patients with advanced CHF or CKD often have increased Ang II levels and cachexia, and angiotensin-converting enzyme inhibitor treatment improves weight loss. It was found that Ang II infusion in rodents leads to skeletal muscle wasting. Ang II increases cytokines and circulating hormones, such as tumor necrosis factor-α, interleukin-6, serum amyloid-A and glucocorticoids, which regulate muscle protein synthesis and degradation. Ang II-induced muscle wasting is caused by alterations in insulin-like growth factor-1 signaling, enhanced muscle protein breakdown via the ubiquitin-proteasome system and decreased appetite resulting from the downregulation of hypothalamic orexigenic neuropeptides, such as Npy and orexin. Ang II also inhibits 5' adenosine monophosphate-activated protein kinase activity and disrupts normal energy balance via the activation of 5' adenosine monophosphate-activated protein kinase phosphatase PP2Cα. Furthermore, Ang II inhibits skeletal muscle stem (satellite) cell proliferation, leading to lowered muscle regenerative capacity. Distinct satellite cell angiotensin receptor subtypes have different effects on different stages of differentiation and are critical for the regulation of muscle regeneration. These data suggest that the renin-angiotensin system plays a critical role in mechanisms underlying cachexia in chronic disease states, and it is a promising target for the treatment of muscle atrophy in patients with diseases such as CHF and CKD.

cAMP-dependent activation of protein kinase A attenuates respiratory syncytial virus-induced human airway epithelial barrier disruption

PubMed Central

Harford, Terri J.; Linfield, Debra T.; Altawallbeh, Ghaith; Midura, Ronald J.; Ivanov, Andrei I.; Piedimonte, Giovanni

2017-01-01

Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC proteins by confocal microscopy. Increased intracellular cAMP level significantly attenuated RSV-induced disassembly of AJC. These barrier-protective effects of cAMP were due to the activation of PKA signaling and did not involve Epac activity. Increased cAMP level reduced RSV-induced reorganization of the actin cytoskeleton, including apical accumulation of an essential actin-binding protein, cortactin, and inhibited expression of the RSV F protein. These barrier-protective and antiviral-function of cAMP signaling were evident even when cAMP level was increased after the onset of RSV infection. Taken together, our study demonstrates that cAMP/PKA signaling attenuated RSV-induced disruption of structure and functions of the model airway epithelial barrier by mechanisms involving the stabilization of epithelial junctions and inhibition of viral biogenesis. Improving our understanding of the mechanisms involved in RSV-induced epithelial dysfunction and viral pathogenesis will help to develop novel anti-viral therapeutic approaches. PMID:28759570

Relationships of methacholine and adenosine 5'-monophosphate (AMP) responsiveness to the postbronchodilator FEV₁/FVC ratio in children with asthma.

PubMed

Suh, Dong In; Choi, Sun Hee; Lee, Ju Kyung; Kim, Jin-Tack; Koh, Young Yull

2011-05-01

Airway remodeling has been assumed to cause bronchial hyperresponsiveness (BHR). A low postbronchodilator FEV₁/FVC ratio has been suggested to be a functional surrogate marker of airway remodeling in asthma. BHR is commonly assessed by bronchial challenges using direct or indirect stimuli. The aim of this study was to compare BHR to methacholine and adenosine 5'-monophosphate (AMP) with regard to their relationship with a marker of airway remodeling in children with asthma. Methacholine and AMP challenge tests were performed in 129 children with asthma, aged 12 years, and a provocative concentration causing a 20% fall in FEV₁ (PC₂₀) was calculated for each challenge. All subjects also underwent pre- and postbronchodilator spirometry. A postbronchodilator FEV₁/FVC ratio below the lower limits of normal was used as a marker of airway remodeling. A low postbronchodilator FEV₁/FVC ratio was found in 17 subjects (13.2%). These subjects had a significantly lower methacholine PC₂₀ (geometric mean: 0.63 mg/mL, range of 1 SD: 0.17-2.29) than those (n = 112) with a normal postbronchodilator FEV₁/FVC ratio (2.42 mg/mL, 0.57-10.32, p = .000), whereas AMP PC₂₀ was similar between the two groups (22.1 mg/mL, 3.9-125.9 vs. 27.7 mg/mL, 4.2-183.5, p = .231). In the whole group of subjects, methacholine PC₂₀, but not AMP PC₂₀, correlated significantly with the postbronchodilator FEV₁/FVC ratio (r = 0.340, p = .000, and r = 0.056, p = .526, respectively). Our results provide evidence, though indirect, that BHR to methacholine is related to airway remodeling in children with asthma and suggest that BHR to methacholine may be a better marker of airway remodeling than BHR to AMP.