Adenine Nucleotide Content Research Articles

Adenine nucleotide content and activity of AMP catabolism enzymes in the kidney of rats fed on diets with different protein and sucrose content

Background. Excessive consumption of sucrose or protein deficiency in the diet can induce metabolic disorders in the kidney, whose functioning requires significant ATP energy expenditure. The study investigated the levels of the purine nucleotides ATP, ADP, AMP, and the activity of the enzymes FoF1-ATPase, 5′-nucleotidase, and AMP deaminase in the kidneys of rats exposed to different levels of protein and sucrose in their diet. Materials and Methods. The research was conducted on white non-linear rats, which were kept under different dietary regimens for a period of 4 weeks. Quantitative evaluation of the ATP, ADP, and AMP content was performed by thin-layer chromatography on Silufol sheets. FoF1-ATPase activity was determined by the accumulation of Pi. 5′-nucleotidase activity was measured based on the amount of inorganic phosphorus released in AMP hydrolysis. AMP deaminase activity was determined by the accumulation of ammonia. Results and Discussion. Research results revealed that in the mitochondria of the animals’ kidneys under conditions of low-protein diet, a significant reduction in AMP content was observed compared to the control, while ATP and ADP content remained unchanged. Simultaneously, the activities of 5′-nucleotidase, AMP deaminase, and FoF1-ATPase in the kidneys of animals on a low-protein diet were maintained at control levels. However, in rats maintained on a low-protein/high-sucrose diet, depletion of all adenine nucleotides is observed against an increase in the hydrolytic activity of FoF1-ATPase, AMP deaminase, and 5′-nucleotidase activities. The 5′-nucleotidase activity in animals of this group reaches maximum values in comparison with the control, indica­ting an enhanced AMP degradation mediated by 5′-nucleotidase in the conditions of low-protein/high-sucrose diet consumption. Conclusion. Excessive sucrose consumption in the context of dietary protein deficiency is accompanied by a depletion of the adenine nucleotides pool in the mitochondrial fraction and a significantly increased activity of purine catabolism enzymes in the cytosolic fraction of rat kidneys. This may result in an imbalance in the energy supply of renal cells. The obtained results open up prospects for developing a strategy for correc­ting energy metabolism disorders in the conditions of nutritional imbalance.

Investigating ZMP Accumulation in C2C12 Myotubes with AICAR and Prodrug-39

5’Adenosine monophosphate (AMP) is a signaling molecule and a known regulator of enzymes in glycolysis, glycogen breakdown, and may play a role in regulating protein synthesis. It is also a known activator of AMP-dependent protein kinase (AMPK), a key energy sensor within the cell. Activation of AMPK has been linked to the regulation of mitochondrial biogenesis, upregulation of BCAA catabolism, and increased skeletal muscle glucose uptake and fatty acid oxidation. AMPK can be activated pharmacologically with 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an adenosine analogue. AICAR enters the cell through adenosine transporters and undergoes phosphorylation by Adenosine Kinase forming AICAR monophosphate (ZMP). Unfortunately, the poor bioavailability of AICAR limits its use pharmacologically. Prodrug-39 (P39) is comprised of ZMP with a 4-nitrophenol group to allow membrane permeability and subsequent ZMP formation without the need of membrane transporters. We investigated the ZMP accumulation in C2C12 myotubes following exposure to different concentrations of P39 or AICAR. Based on preliminary data, we hypothesized that AICAR treatments would be more effective at producing ZMP over short periods of time and when treatment concentrations were high, but that P39 treatments would result in greater and more consistent ZMP accumulation over longer periods of time when treatment concentrations were low. We first measured the absorbance of 4-nitrophenol (4NP) within the cell media to determine phosphodiesterase cleavage of P39. Nitrophenol absorbance was 3.9 times higher at 1000 μM compared to 100 μM treatment of P39 after 15 minute incubation, and 13.3 times higher at 11 hours. The nitrophenol absorbance per hour increased linearly between 250 μM to 1000 μM of P39. Adenine nucleotide content and ZMP accumulation in treated cells was determined following organic protein extraction and UV-Vis ultra-performance liquid chromatography (UPLC). After 13 hours of treatment with 500 μM AICAR or P39, ZMP accumulation was 14 fold higher following AICAR treatment compared to P39 (59.7±5.0 vs 4.2±0.1 μmol/g protein). Interestingly, ZMP accumulation was not different between 100 μM treatments (5.6±1.3 vs. 5.7±1.5 μmol/g protein). This suggests that phosphodiesterase cleavage of P39 is likely the limiting factor for ZMP accumulation. Interestingly, while ZMP accumulation following 13 hour incubation with 100 μM AICAR or P39 was not different, ATP content was approximately 50% higher in P39 treated cells (33.7±5.8 vs. 51.0±5.8 μmol/g protein). While the measurement of ZMP accumulation at very low treatment concentrations (10 μM) is very diffcult, P39 exposure for 1 to 1.5 hours resulted in a 3 to 8 fold increase in ZMP content compared to AICAR. This data illustrates the potential for P39 as a drug capable of accumulating greater ZMP than AICAR at very low treatment concentrations, however the capacity of phosphodiesterase activity may limit the application of this drug. This work was funded by a grant from Skylark Biosciences. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Red Blood Cell Adenylate Energetics Is Related to Endothelial and Microvascular Function in Long COVID.

Adenine nucleotides play a critical role in maintaining essential functions of red blood cells (RBCs), including energy metabolism, redox status, shape fluctuations and RBC-dependent endothelial and microvascular functions. Recently, it has been shown that infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might lead to morphological and metabolic alterations in erythrocytes in both mild and severe cases of coronavirus disease (COVID-19). However, little is known about the effects of COVID-19 on the nucleotide energetics of RBCs nor about the potential contribution of nucleotide metabolism to the long COVID syndrome. This study aimed to analyze the levels of adenine nucleotides in RBCs isolated from patients 12 weeks after mild SARS-CoV-2 infection who suffered from long COVID symptoms and to relate them with the endothelial and microvascular function parameters as well as the rate of peripheral tissue oxygen supply. Although the absolute quantities of adenine nucleotides in RBCs were rather slightly changed in long COVID individuals, many parameters related to the endothelial and microcirculatory function showed significant correlations with RBC adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentration. A particularly strong relationship was observed between ATP in RBCs and the serum ratio of arginine to asymmetric dimethylarginine-an indicator of endothelial function. Consistently, a positive correlation was also observed between the ATP/ADP ratio and diminished reactive hyperemic response in long COVID patients, assessed by the flow-mediated skin fluorescence (FMSF) technique, which reflected decreased vascular nitric oxide bioavailability. In addition, we have shown that patients after COVID-19 have significantly impaired ischemic response parameters (IR max and IR index), examined by FMSF, which revealed diminished residual bioavailability of oxygen in epidermal keratinocytes after brachial artery occlusion. These ischemic response parameters revealed a strong positive correlation with the RBC ATP/ADP ratio, confirming a key role of RBC bioenergetics in peripheral tissue oxygen supply. Taken together, the outcomes of this study indicate that dysregulation of metabolic processes in erythrocytes with the co-occurring endothelial and microvascular dysfunction is associated with diminished intracellular oxygen delivery, which may partly explain long COVID-specific symptoms such as physical impairment and fatigue.

Evolution of High-Affinity ssDNA Sequence to Carbon Nanotube

Colloidal ssDNA-wrapped single walled carbon nanotube (ssDNA-SWCNT) has been used as the second near-infrared (NIR-II, 1000 -1700 nm) fluorescent nanoprobes with outstanding biocompatibility, and acts as various optical nano sensors for biological analytes such as a dopamine and serotonin. The stability and affinity of ssDNA on the SWCNT surface significantly depends on the sequence of ssDNA, however the large-scale quantitative analysis between ssDNA sequence library, which has high affinity to the SWCNT surface, was repeatedly selected from a random ssDNA(N30) library (~1018) and the resulting sequence data was analyzed. The binding affinity of ssDNA to SWCNT was quantitatively characterized. To quantitatively analyze the kinetics of the process which occurs solvatochromic shift by sodium cholate, assumed the dynamics follow a first-order kinetic model and fit the experimental data using exponential regression, from which decay time constants were determined. As a result, the highest binding affinity of ssDNA to SWCNT was quantitatively characterized by time constant (1.5 X106 sec) which increased by 100 times compared to the N30 random library. Through analysis of the ssDNA sequences with high affinity for the SWCNT surface, we found that the sequences contain high contents of cytosine and adenine nucleotides and complementary base pairs appeared at distance of 5 to 8 bases. This novel sequence library can be applied for the preparation of the ultra-stable ssDNA-SWCNT dispersion and used for anchoring surface ligands for SWCNT as biosensor. Figure 1

Electrolyzed reduced water could regulate muscular energy producing ability of heat-exposed broiler chickens

We have previously shown that alleviation in ROS-induced oxidative damage to skeletal muscle and improvement in production efficiencies are observed in broiler chickens supplied with electrolyzed-reduced water when exposed to 5 days-long chronic heat exposure. In this study, we investigated whether this reduced water may also improve those parameters in broiler chickens exposed to 14 days-long chronic heat exposure. Broiler chickens (Gallus gallus) were fed either a control diet with tape water or control diet supplied with electrolyzed-reduced water for 14 d after hatch, and then exposed either to heat stress (34oC for 14 days), or kept at a thermoneutral temperature (24oC). Skeletal muscle substrate metabolism (3HADH and CS activities), adenine nucleotides concentration, enzymatic scavenging systems were studied. We confirmed that heat-stressed broilers decreased weight gain and feed consumption compared to control broilers. In contrast, heat-exposed broilers supplied with electrolyzed-reduced water significantly and definitely improved performance when compared to heat-stressed control broilers. On exposure to long-term heat treatments, broiler supplied with reduced water showed higher CS activity, indicating that the reduced water could improve energy-producing ability via modulating TCA cycle. We also found that the reduced water could definitely improve the total adenine nucleotide (TAN), adenylated energy charge (AEC), and ATP to ADP ratios under such conditions. On the other hand, broiler chickens supplied with reduced water kept relatively constant the SOD, catalase, and GPx activities, implying that reduced water could support the enzymatic scavenging systems. Taken together, these results suggest that broiler chickens supplied with electrolyzed-reduced water could improve energy producing ability via modulating TCA-cycle enzyme, and lead to improving performance.

Mechanisms of regenerative preconditioning in pigs with subtotal hepatectomies.

Portal vein occlusion shortly before extended hepatic resections has hepatoprotective properties, but its molecular effects have not been elucidated. We characterized the impact of regenerative preconditioning by portal vein embolization (PVE) on hepatic energy metabolism and cytokine expression. About 90% hepatectomies were performed in normal pigs (Control) and in pigs that underwent a PVE 24 h before the surgery (n = 10/group). Blood biochemistry and coagulation, liver damage, liver function (ICG), hepatic content of adenine nucleotides, and hepatic expression of inflammatory mediators (RT-PCR and WB) were determined before the hepatectomy, 15 min, and 24 h later. All PVE and hepatectomies were successfully accomplished. The 90% hepatectomy resulted in: Immediate reduction of ATP, leading to persistent decreases of energy load and ATP/ADP ratio up to the 24-h time-point; and pro-inflammatory expression profile of cytokines in the remnant liver. Prior performance of PVE attenuated the bioenergetic alterations and prevented many of the changes in hepatic cytokine expression. Regenerative preconditioning by PVE improved hepatic energy metabolism and modulated inflammatory mediators in the remnant liver in pigs undergoing major hepatectomies, potentially contributing to its hepatoprotective effects.

Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP

BackgroundSkeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1. MethodsThe modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin. ResultsThe study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1. ConclusionsThe desensitization to ATP is due to the modification of His-51 of the Zn2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized. General significanceThe progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution.

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle

BackgroundSkeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. MethodsAMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. ResultsTA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption (−15%). ConclusionsIncreased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

Reproductive senescence impairs the energy metabolism of human luteinized granulosa cells

Research questionFemale age is the single greatest factor influencing reproductive performance and granulosa cells are considered as potential biomarkers of oocyte quality. Is there an age effect on the energy metabolism of human mural granulosa cells? DesignObservational prospective cohort and experimental study including 127 women who had undergone IVF cycles. Women were allocated to two groups: a group of infertile patients aged over 38 years and a control group comprising oocyte donors aged less than 35 years. Individuals with pathologies that could impair fertility were excluded from both groups. Following oocyte retrieval, cumulus and granulosa cells were isolated and their bioenergetic properties (oxidative phosphorylation parameters, rate of aerobic glycolysis and adenine nucleotide concentrations) were analysed and compared. ResultsHuman mural luteinized granulosa and cumulus cells present high rates of aerobic glycolysis that cannot be increased further when mitochondrial ATP synthesis is inhibited. Addition of follicular fluid to the experimental media is necessary to reach the full respiratory capacity of the cells. Granulosa cells from aged women present lower mitochondrial respiration (12.8 ± 1.6 versus 11.2 ± 1.6 pmol O2/min/mg; P = 0.046), although mitochondrial mass is not decreased, and lower aerobic glycolysis, than those from young donors (12.9 ± 1.3 versus 10.9 ± 0.5 mpH/min/mg; P = 0.009). The concurrent decrease in the two energy supply pathways leads to a decrease in the cellular energy charge (0.87 ± 0.01 versus 0.83 ± 0.02; P < 0.001). ConclusionsHuman mural luteinized granulosa cells exhibit a reduction in their energy metabolism as women age that is likely to influence female reproductive potential.

Metabolic regulation in mitochondria as a prospective way of body rejuvenation

Scientists around the world are looking for ways to achieve high-quality and active longevity. The complexity of these searches is associated with the lack of unified concept of aging mechanisms, the problem of extrapolating the results of studies of geroprotectors from model organisms to humans. Therefore, modern medicine can only slow down the aging process so far. Nevertheless, it has been proven that disturbances in the functioning of mitochondria are an important link in the pathogenesis of aging; therefore, regulation of their metabolism is a promising way of rejuvenating the body. A decrease in protein synthesis and a general decrease in the intensity of the oxidative enzymes action are interrelated. Energy production for the protein and DNA synthesis decreases with a decrease in the intensity of oxidative processes. Dysfunction of mitochondrial adaptive reactions also develops with age, which may be associated with the low efficiency of the NADH dehydrogenase complex – NADH ubiquinone oxidoreductase, which catalyzes the transfer of electrons from NADH to ubiquinone/coenzyme Q10, resulting in a decrease in electron transport and an increase of active oxygen forms. Coenzyme Q10 reducing plays a very important role in the aging process, as it is an antioxidant and, unlike other antioxidants, is produced in the body. An increase in mitochondrial biogenesis, as well as an increase in the rate and efficiency of mitochondrial metabolism (with a calorie restricted diet for example) helps to improve the energy supply of cells and prolong life.A drug based on xylitol Xylate can be a source of triglycerophosphate by metabolism through the pentose phosphate cycle and thereby reduce the amount of free fatty acids that can be oxidized into acetyl-CoA, increases the content of adenosine triphosphate acid and other adenine nucleotides in liver, and has a pronounced antiketogenic effect. It can reduce intoxication and improve microcirculation, which is especially important for high-quality and active longevity.Activation of mitochondrial metabolism is a promising direction in the aging prevention, since it allows balancing metabolic processes not only in cells, but also in body as a whole.

Sequential Changes of Total Adenine Nucleotide and Adenylic-Acid Energy Charge in Muscles of Rats after Death.

Objective To determine the purine adenylate [adenosine triphosphate （ATP）, adenosine diphosphate （ADP）, adenosine monophosphate （AMP）] content in the muscles of both hind limbs of rats at different postmortem interval （PMI）, calculate the changes in the total adenine nucleotide （TAN） content and the adenylic-acid energy charge （AEC）, and explore their relationship with PMI. Methods Healthy rats were sacrificed by cervical dislocation and kept at 20 ℃. The muscles of their hind limbs were extracted at 0, 24, 48, 72, 96, 120, 144, and 168 h after death. Reversed-phase high performance liquid chromatography was used to determine the content of purine adenylates, the TAN and AEC of the muscles of the both hind limbs were calculated, and the related regression equations of their relationship with PMI were established. Results Within 168 h of death of rats, the trend of ATP change was different from ADP, and the content of AMP continuously increased. The TAN value gradually increased with the extension of PMI, and the AEC showed a downward trend within 168 h after death. Among them, the patterns of AEC changes with PMI were obvious, the correlation coefficient was high （ R2=0.903）, and the curve fitting relationship was good; the fitting relationship between ATP, ADP, AMP, TAN and PMI was poor （ R2=0.198-0.754）. Conclusion The postmortem change patterns of AEC provide new research ideas for PMI estimation in the forensic field.

Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.

A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPβS) in a 1–1000 μM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100–1000 μM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 μM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.

Increased AMP deaminase activity decreases ATP content and slows protein degradation in cultured skeletal muscle

BackgroundProtein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. MethodsTo determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. ResultsOverexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. ConclusionsAltogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.

Growth, osmoregulatory response, adenine nucleotide contents, and liver transcriptome analysis of steelhead trout (Oncorhynchus mykiss) under different salinity acclimation methods

Chinese researchers have been working salmon mariculture far offshore in the Yellow sea, and steelhead trout is one of the popular Salmonidae. This study was conducted to investigate the effect of different salinity acclimation methods on the growth, osmoregulation, energy balance, and liver transcriptome of steelhead trout (Oncorhynchus mykiss). Fish (initial weight, 394.50 g) were reared for 40 days after undergoing salinity acclimation at three rates: an abrupt increase in salinity from zero to 30 ppt (T30); an abrupt increase in salinity to 14 ppt, followed by a daily increase of six ppt (T6) or two ppt (T2), until reaching 30 ppt; and no salinity exposure (control group, T0). Subsequently, serum was collected to determine osmolality, iron concentration, Na+-K+-ATP (NKA) and Ca2+-Mg2+-ATP (CMA) activity; muscle and gill samples were collected for the determination of adenosine 5′-triphosphate (ATP); liver transcriptome was compared between the T0 and T2 groups. The final weight, specific growth rate, food conversion efficiency, and the ATP content of fish in the T2 group were significantly higher than those in the other groups. However, the serum osmolality, serum ion concentration, and gill NKA and CMA activity in the T2 group was significantly lower than that in the T6 and T30 groups. A total of 53,679 unigenes were produced, and 1438 differently expressed genes were selected by DESeq. Among them, 587 genes were up-regulated, while 581 were down-regulated. These differential genes were mainly related to the function of metabolites and ions transportation, energy metabolism, signal transduction, immune responses, and structural assembly. The results indicate that steelhead trout with a weight of approximately 400 g is suitable for mariculture, and T2 was the best transition method for salinity acclimation.

Effects of histidine load on ammonia, amino acid, and adenine nucleotide concentrations in rats.

The unique capability of proton buffering is the rationale for using histidine (HIS) as a component of solutions for induction of cardiac arrest and myocardial protection in cardiac surgery. In humans, infusion of cardioplegic solution may increase blood plasma HIS from ~ 70 to ~ 21,000µM. We examined the effects of a large intravenous dose of HIS on ammonia and amino acid concentrations and energy status of the body. Rats received 198mM HIS intravenously (20ml/kg) or vehicle. Samples of blood plasma, urine, liver, and soleus (SOL) and extensor digitorum longus (EDL) muscles were analysed at 2 or 24h after treatment. At 2h after HIS load, we found higher HIS concentration in all examined tissues, higher urea and ammonia concentrations in blood and urine, lower ATP content and higher AMP/ATP ratio in the liver and muscles, higher concentrations of almost all examined amino acids in urine, and lower glycine concentration in blood plasma, liver, and muscles when compared with controls. Changes in other amino acids were tissue dependent, markedly increased alanine and glutamate in the blood and the liver. At 24h, the main findings were lower ATP concentrations in muscles, lower concentrations of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) in blood plasma and muscles, and higher carnosine content in SOL when compared with controls. It is concluded that a load of large HIS dose results in increased ammonia levels and marked alterations in amino acid and energy metabolism. Pathogenesis is discussed in the article.

Impaired l-arginine metabolism marks endothelial dysfunction in CD73-deficient mice

Changes in the ecto-5′-nucleotidase activity—an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and l-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73−/− (CD73−/−) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73−/− mice age groups were characterized by reduced l-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73−/− mice lungs in comparison to EC isolated from WT lungs. The l-Arginine/ADMA ratio in the CD73−/− decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73−/− mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73−/− compared to controls. Blood (erythrocyte) Adenosine-5′-triphosphate and Adenosine-5′-diphosphate levels were reduced in favor to higher blood Adenosine-5′-monophosphate concentration in CD73−/− mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired l-arginine metabolism. CD73 activity seems to protect endothelium.

Resveratrol improves mitochondrial energetic pathway and endothelial function in type 2 diabetic female rat heart submitted to ischemia-reperfusion injury

Type 2 diabetic women present high risk of mortality by cardiovascular (CV) disease in comparison to non-diabetic women. Most of anti-diabetic treatments do not decrease patient's CV mortality, therefore new approaches need to be found. Resveratrol (RSV) is a polyphenol found in grapes reported to be beneficial on oxidative stress, endothelial dysfunction and inflammation involved in CV complications of T2D. We have investigated the effect of RSV on the tolerance to ischemia-reperfusion (IR) injury of type 2 diabetic female Goto-Kakizaki (GK) rat hearts. Four groups were used: a control group, a GK group, a GK group with placebo and a GK group with RSV treatment at the dose of 1mg/kg/day in drinking water for 8 weeks. After 8 weeks of RSV treatment, tolerance to IR injury was assessed during ex vivo experiments, with simultaneous measurement of energy metabolism by 31P MRS, myocardial function and coronary flow. At the end of experiments, PCr, creatine, adenine nucleotides contents and citrate synthase activity were assessed in freeze-clamped hearts as well as nitric oxide and sirtuin pathways. Type 2 diabetic GK rats exhibited cardiac hypertrophy and lower myocardial tolerance to IR injury in comparison to controls. RSV attenuated cardiac hypertrophy and increased myocardial tolerance to IR injury by improving cardiac function, mitochondrial energetic and endothelial pathways. Interestingly, RSV increased ATP, PCr, creatine, total adenine nucleotide heart contents, citrate synthase activity, as well as eNOS, P-Akt and SIRT1 protein expressions in GK rat hearts. RSV induced cardioprotection against IR injury in type 2 diabetic female rats. Thus, RSV presents high potential for preventing and treating CV complications of type 2 diabetic women.

Protective efficacy of dinitrosyl iron complexes with reduced glutathione in cardioplegia and reperfusion.

Disturbed homeostasis of nitric oxide (NO) is one of the causes of myocardial ischemia/reperfusion (I/R) injury during open-heart surgery. This study was designed to explore mechanisms of action of dinitrosyl iron complexes with reduced glutathione ({(GS-)2Fe+(NO+)2}+, DNIC-GS) added to crystalloid cardioplegia or reperfusion solution in isolated working rat hearts. Hearts of male Wistar rats were subjected to cardioplegic arrest by St. Thomas' Hospital cardioplegic solution (STH) and normothermic global ischemia followed by reperfusion. DNIC-GS were used with STH or during early reperfusion. Lactate dehydrogenase (LDH) activity in the coronary effluent and myocardial contents of adenine nucleotides, phosphocreatine, and lactate were determined spectrophotometrically. Reactive oxygen species (ROS) formation in the coronary effluent and myocardial DNIC content was assessed by EPR technique. Cardioplegia or reperfusion with DNIC-GS significantly improved recovery of coronary flow and cardiac function compared with control. Carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide] (C-PTIO), a selective NO scavenger, reduced/abolished protective action of DNIC-GS. Enhanced recovery of cardiac function with DNIC-GS reduced LDH release in the coronary effluent, augmented recovery of myocardial energy state, and decreased formation of ROS-generating systems at reperfusion. Beneficial effects of DNIC-GS were related to the transfer of [Fe(NO)2] cores to thiol groups of myocardial proteins to form intracellular DNIC pools. The study concluded that DNIC-GS is a promising adjunct agent for metabolic and antioxidant protection of the heart during cardioplegic arrest and reperfusion.

Cardiometabolic efficacy and toxicological evaluation of a pharmacological galanin receptor agonist

The goal of this study was to examine effects of a novel galanin receptor agonist GalR1-3 [bAla14, His15]-galanine 2-15 (G), obtained by automatic solid-phase synthesis, on the metabolic state of the area at risk and the size of acute myocardial infarction (MI) in rats in vivo and evaluate its toxicity in BALB /c mice. In anesthetized rats, regional ischemia was simulated by coronary artery occlusion and then coronary blood flow was restored. The peptide G was administered intravenously (i.v.) with a bolus after a period of regional ischemia in the dose range of 0.25-3.0 mg/kg. The sizes of MI and the activities of creatine kinase-MB (СK-MB) and lactate dehydrogenase (LDH) in blood plasma were estimated. The effect of administration of the optimal dose of G (1.0 mg/kg) on myocardial content of adenine nucleotides (AN), phosphocreatine (PCr), creatine (Cr) and lactate was studied. I.v. administration of G to rats at a dose of 1.0 mg/kg slightly affected hemodynamic parameters, but reduced MI size by 40% and decreased plasma LDH and CK-MB activity by the end of reperfusion compared to control. These effects were accompanied by a significant improvement in energy state of area at risk (AAR) - an increase in myocardial content of ATP, åAN, PCr and åCr, and combined with a decrease in myocardial lactate level compared with the control. Toxicity of peptide G was studied with a single intraperitoneal injection of 0.5-3.0% solution of the peptide substance to mice. The absence of signs of intoxication and death of animals after G injection in the maximum possible dose did not allow determining the value of the average lethal dose. The results indicate therapeutic potential of the peptide G for preventing myocardial ischemia and reperfusion injury and feasibility for further study of its pharmacological properties and mechanisms of action.

The effects of adenine nucleotide perfusion on interstitial adenosine production in rat skeletal muscle.

The purpose of the present study was to utilize the microdialysis technique in rat skeletal muscle to perfuse varying concentrations of AMP, ADP, and ATP into the interstitium to examine the effects that these adenine nucleotides have on the production of adenosine in the interstitial space. Interstitial adenosine production appears to be related to the type (ATP, ADP, or AMP) and concentration (2-60 μmol/L) of the adenine nucleotide perfused. Interstitial adenosine levels increased (P < 0.05) from baseline (0.18 ± 0.02 and 0.22 ± 0.02 μmol/L) to 0.23 ± 0.02 and 0.41 ± 0.05 μmol/L following 5 and 30 μmol/L AMP perfusion, respectively. Similarly, perfusion with 30 μmol/L ADP and 30, 40, and 60 μmol/L ATP resulted in an increase (P < 0.05) in interstitial adenosine concentration from baseline (0.25 ± 0.02, 0.26 ± 0.02, 0.19 ± 0.03, and 0.14 ± 0.02 μmol/L) to 0.30 ± 0.02, 0.32 ± 0.02, 0.36 ± 0.04, and 0.33 ± 0.04 μmol/L, respectively. Interestingly, the most prominent increase in interstitial adenosine production occurred during the perfusion of 60 μmol/L ATP (126% increase from baseline). These data strongly suggest that interstitial ATP may play a more potent role in stimulating interstitial adenosine production as compared with ADP or AMP. In addition, interstitial adenosine production can occur independent of muscle contraction (voluntary or involuntary) or hypoxia when adequate concentrations of adenine nucleotides are available.