Total Adenosine Nucleotides Research Articles

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

β2-Adrenoreceptor Agonist Inhalation During Ex Vivo Lung Perfusion Attenuates Lung Injury

Attenuation of ischemia reperfusion injury (IRI) is important in lung transplantation. Our group previously reported that β2-adrenoreceptor agonist inhalation during the period before procurement successfully attenuated IRI in donated lungs after cardiac death. We therefore hypothesized that β2-adrenoreceptor agonist inhalation during exvivo lung perfusion (EVLP) after procurement might also have a protective effect. Cardiac-dead beagles were left at room temperature for 210 minutes, and all lungs were subsequently procured and subjected to EVLP for 240 minutes. The beagles were allocated to 2 groups: the β2 group (receiving an aerosolized β2-adrenoreceptor agonist 20 minutes after initiation of EVLP; n= 7) and the control group (receiving an aerosolized control solvent at the same time point; n= 6). Physiologic data, including lung function, were evaluated during EVLP. The β2 group showed significantly lower peak airway pressure and pulmonary artery pressure than the control group. Dynamic pulmonary compliance was higher, pulmonary vascular resistance (PVR) was lower, and the wet-to-dry lung weight ratio was lower in the β2group than in the control group. Cyclic adenosine monophosphate (cAMP) and total adenosine nucleotide (TAN) levels in lung tissue after EVLP were higher in the β2 group than in the control group. The β2 group also showed more cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. After procurement, β2-adrenoreceptor agonist inhalation during EVLP attenuates lung injury in a canine model of organ donation after cardiac death.

A 4-week consumption of medium roast and dark roast coffees affects parameters of energy status in healthy subjects

ScopeThis study intended to clarify whether two coffee brews, a market blend (MB) and a study blend (SB), containing equal amounts of caffeine, but differing in their contents of N-methylpyridinium, trigonelline and chlorogenic acids, differentially affect blood lipid profiles and glucose concentrations as well as blood platelet phosphodiesterase and lymphocyte energy charge potential in healthy volunteers. Methods and resultsIn this double-blinded, randomized, controlled cross-over intervention study, 84 healthy normal-weight female and male volunteers consumed 750mL of medium roast MB and dark roast SB coffee per day for 4weeks. Following MB and SB coffee intervention, plasma free fatty acid concentrations equally increased (p<0.001) by 140±187μM and 178±160μM, respectively. Plasma glucose remained largely unchanged. Lymphocyte adenosine nucleotide analysis revealed a comparable rise in the energy charge potential, as calculated from ADP/ATP concentrations, adjusted to total adenosine nucleotides. Blood platelet phosphodiesterase activity was found decreased to about the same extent (p<0.001). Levels of HDL-cholesterol, adiponectin, leptin, insulin and osteopontin were found to some extent differentially influenced by MB, respectively SB coffee. ConclusionThe results of this intervention study indicate that MB and SB coffees, although differing in contents of N-methylpyridinium, trigonelline and chlorogenic acids, largely exert similar biological effects as monitored by the biomarkers tested.

Mutation of mitochondrial ATP8 gene improves hepatic energy status in a murine model of acute endotoxemic liver failure

Aims Mitochondria not only generate and modulate bioenergy but also serve as biosensors for oxidative stress, and eventually become effector organelles for cell viability. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene affects the course of endotoxemic acute liver failure. Main methods C57BL/6J ( ATP8 wild type) and C57BL/6J-mt FVB/N ( ATP8 mutant) mice were challenged with d-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) for induction of acute liver failure, and studied 6 h thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and levels of hepatocellular apoptosis, hepatic adenosine nucleotides and oxidative stress. Additionally, survival rates were assessed. Key findings Induction of endotoxemic liver failure provoked marked liver damage, which was coexistent with a drop of total adenosine nucleotide levels and increased oxidative stress. Of interest, oxidative stress was higher in the GalN/LPS challenged ATP8 mutants compared to wild types. Concomitantly, adenosine triphosphate (ATP) levels in livers of mice carrying the ATP8 mutation remained higher than those in wild type mice. As net result, ATP8 mutants showed lower transaminase release and a tendency to better survival rate upon GalN/LPS exposure compared to wild types. Significance Our findings demonstrate that mutation in the ATPase subunit-8 partially protects mice against endotoxemic stress, most probably due to better hepatic energy status despite elevated oxidative stress. Thus, modulating mitochondrial function to preserve bioenergetic status may be an effective strategy to protect against sepsis-induced multiorgan dysfunction.

Proteomic and metabolomic analysis of cardioprotection: Interplay between protein kinase C epsilon and delta in regulating glucose metabolism of murine hearts

We applied a combined proteomic and metabolomic approach to obtain novel mechanistic insights in PKCɛ-mediated cardioprotection. Mitochondrial and cytosolic proteins from control and transgenic hearts with constitutively active or dominant negative PKCɛ were analyzed using difference in-gel electrophoresis (DIGE). Among the differentially expressed proteins were creatine kinase, pyruvate kinase, lactate dehydrogenase, and the cytosolic isoforms of aspartate amino transferase and malate dehydrogenase, the two enzymatic components of the malate aspartate shuttle, which are required for the import of reducing equivalents from glycolysis across the inner mitochondrial membrane. These enzymatic changes appeared to be dependent on PKCɛ activity, as they were not observed in mice expressing inactive PKCɛ. High-resolution proton nuclear magnetic resonance ( 1H-NMR) spectroscopy confirmed a pronounced effect of PKCɛ activity on cardiac glucose and energy metabolism: normoxic hearts with constitutively active PKCɛ had significantly lower concentrations of glucose, lactate, glutamine and creatine, but higher levels of choline, glutamate and total adenosine nucleotides. Moreover, the depletion of cardiac energy metabolites was slower during ischemia/reperfusion injury and glucose metabolism recovered faster upon reperfusion in transgenic hearts with active PKCɛ. Notably, inhibition of PKCɛ resulted in compensatory phosphorylation and mitochondrial translocation of PKCδ. Taken together, our findings are the first evidence that PKCɛ activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCδ and PKCɛ in cardioprotection.

Abstract 511: Mitochondrial Complex I -Regulated Alteration of Carbohydrate Oxidation and Energy Metabolites Reduces Ischemia-reperfusion Injury in Hearts from Mice Exposed to Moderate Ethanol Intake

Background: Moderate alcohol consumption is cardioprotective in large part through mechanisms that converge on mitochondria. As a primary entry point for reducing equivalents into the respiratory chain, the state of complex I activation has important consequences for overall mitochondrial function and cell homeostasis. Hypothesis : Complex I is a key control point in regulation of carbohydrate oxidation and energy production that influence the extent of cardiac injury in hearts from ethanol-fed animals subjected to ischemia-reperfusion (I/R). Methods and Results: Isolated hearts from C57BL/6 mice fed 10% ethanol (vol/vol) as drinking water for 16 weeks (E) and age-matched controls were subjected to 30 min global ischemia and 30 min reperfusion. Hemodynamics showed E hearts had better contractile recovery (left ventricular developed pressure 62±6 vs. 24±5 mmHg) and less cell damage (creatine kinase release 0.32±0.1 vs. 0.65±0.1 U/min/g) than controls after reperfusion. Mitochondrial NADH-Oxidase (1.2±0.2 vs.0.58±0.1 μg/m/mg), NADH-ubiquinone reductase (complex I, 1.04±0.2 vs. 0.48±0.1 μg/m/mg), state 3 oxygen consumption rates (266±28 vs. 152 ± 18 ngAtomO/m/mg), respiratory control ratios (5.2±0.4 vs. 3.1±0.2), aconitase activity (0.73±0.2 vs. 0.48±0.1 μmol/m/mg), and lipid peroxidation (MDA 216±22 vs. 348±14 nmol/g ) were significantly improved in E hearts after I/R compared to control hearts (all P&lt;0.05, n=3–12). EM showed that the severity of mitochondrial ultrastructure deformation in E hearts was attenuated. In addition, high resolution 1H NMR spectroscopy revealed that the myocardial metabolites alanine, glycine, creatine/phosphocreatine, NAD, total adenosine nucleotide, and ATP were better preserved, whereas lactate and succinate were reduced in E heart tissue extracts relative to control hearts. Conclusions: Moderate alcohol consumption reduces myocardial I/R injury. The mechanism of the cardioprotection in part depends on mitochondria -mediated survival pathways. Within the mitochondria, preservation of complex I activity is critical for maintaining overall respiratory function and cardiac energy generation.

Energy and Redox States in the C6 Glioma Cells Following Acute Exposure to Zn, Se+4, and Se+6 and the Correlation with Apoptosis

Changes in cellular energy and redox states in the C6 glioma cells exposed to increasing concentrations of either Zn or Se were studied to examine whether different elements cause different patterns of changes in cellular metabolism. Following a 3-h exposure, both Zn and Se+4 caused dose-dependent decreases in cell viability and total adenosine nucleotides (TAN = ATP + ADP + AMP). In addition, Zn caused a dose-dependent increase in cellular ATP/TAN and a decrease in the ADP/TAN and AMP/TAN. These changes resulted in a significant increase in energy charge potential (ECP = [ATP + 0.5ADP]/TAN). Se+4, on the other hand, caused a dose-dependent decrease in ATP/TAN but an increase in both ADP/TAN and AMP/TAN, resulting in a dose-dependent decrease in ECP. Both Zn and Se+4 caused a dose-dependent decrease in GSH/GSSG and an increase in GSH + GSSG when compared to TAN. In contrast to Zn and Se+4, the nontoxic Se+6 caused no significant changes in cellular energy states but reduced the GSH/GSSG ratio from 3.14 ± 0.49 to 2.05 ± 0.29, which could be explained by the effect of Se on enzymes responsible for GSH metabolism. As the cellular ATP level has been considered an important element that mediates the mode of cell death, it was suggested that a significant increase in ATP/TAN upon exposure to Zn would indicate that cell death occurred via apoptosis, while Se+4 caused a different pattern of cell death. This was confirmed by the appearance of cells with fragmented nucleus in cells treated with Zn, but not Se+4 and Se+6. The results demonstrated that different chemicals caused different patterns of metabolic changes. The correlation between metabolic changes and the mode of cell death was discussed.

Analysis of changes in energy and redox states in HepG2 hepatoma and C6 glioma cells upon exposure to cadmium

The energy and redox states of the HepG2 hepatoma and the C6 glioma cells were studied by quantifying the levels of ATP, ADP, AMP, GSH, and GSSG. These values were used to calculate the energy charge potential (ECP = [ATP + 0.5ADP]/TAN), total adenosine nucleotides (TAN = ATP + ADP + AMP), total glutathione (TG = [GSH + GSSG]/TAN), and the redox state (GSH/GSSG ratio). For comparison between cell types, the level of each energy metabolite (ATP, ADP, and AMP) was normalized against TAN of the respective cell. The results showed that ATP:ADP:AMP ratio was 0.76:0.11:0.13 for the HepG2 cells and 0.80:0.11:0.09 for the C6 glioma cells. ECP was 0.81 +/- 0.01 and 0.85 +/- 0.01 for the HepG2 and the C6 glioma cells, respectively. GSH/GSSG ratio was 2.66 +/- 0.16 and 3.63 +/- 0.48 for HepG2 and C6 glioma cells, respectively. TG was 3.2 +/- 0.54 for the HepG2 cells and 2.43 +/- 0.18 for the C6 glioma cells, indicating that the level of total glutathione is more than two to three times higher than the total energy metabolites in these cell lines. Following a 3-h incubation in medium containing different concentrations of Cd, there was a dose-dependent decrease in cell viability. The 3-h LC50 for the HepG2 cells was 0.5 mM and that for the C6 glioma cells was 0.4 mM. Cellular TAN decreased with a decrease in cell viability. Upon careful analysis of the energy state, there was a significant increase in relative amount of ATP and decrease in ADP and AMP in both cells as Cd concentration increased from 0 to 0.1, 0.2, and 0.6 mM. However, ECP in both cell lines increased, which indicated that the level of high energy phosphate was adequate. There was also a significant increase in TG and a significant decrease in GSH/GSSG in the C6 glioma cells when cells were exposed to as low as 0.1 mM Cd, which suggested that the cellular redox state was compromised. The HepG2 cells, on the other hand, showed no significant change in both TG and GSH/GSSG level until Cd concentration reached 0.6 mM. Results of the present study also showed that there were differences between the two cells in response to the same level of Cd exposure. The C6 glioma cells were more sensitive to Cd-induced injuries. Although there was a decrease in total amount of high energy phosphate as cell viability decreased, the surviving cells were not devoid of high energy phosphates. The relative abundance of ATP amongst the adenosine nucleotide pool and the increase in ECP could be interpreted as a way the cells signal the conservation of energy utilization in response to the damaged mitochondrial function. This move for energy conservation might be the cause of eventual cell death through the process of apoptosis.

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

A method using ion-pairing liquid chromatography–mass spectrometry (MS) was developed for analyzing adenosine 5 ′-monophosphate (AMP), adenosine 5 ′-diphosphate (ADP), and adenosine 5 ′-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization–MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0.1 to 20 μM for AMP, 2 to 20 μM for ADP, and 2.5 to 20 μM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120 μg/ml cause a gradual increased in energy charge potential of the cells.

Modulation of Energy Metabolism in C6 Glioma Cells as Possible Mechanism Contributing to Zinc Neurotoxicity

Elevated tissue levels of zinc (Zn) have been associated with neurodegenerative diseases such as global ischemia, seizure, and Alzheimer's. The mechanism of action of Zn in causing neuronal injury is not clear. One of the possible mechanisms is the ability of Zn to alter cellular energy metabolism. Using the C6 glioma cell as a model, the present study aimed to determined the effects of increasing concentrations of Zn on cellular energy states, as defined by the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP), the total adenosine nucleotides (TAN) (TAN = ATP + ADP + AMP), and the energy charge potential (ECP = [ATP + 0.5 ADP]/TAN). Uptake of Zn was visualized by the appearance of N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide (TSQ)-stained fluorescent granules after a 3-h exposure to Zn in the medium. At [Zn] = 1 mM, cells appeared apoptotic. Levels of ATP and TAN decreased as the level of Zn increased. The change mirrors the increase in cell death as determined by the trypan blue exclusion test. However, when the ratio of ATP:ADP:AMP within the TAN was calculated, the percentage of ATP in the TAN increased significantly, while that of AMP decreased. The change in the relative AMP level mirrored the change in cell viability as measured by the MTT assay, which indicated a decreased in mitochondrial activity. Cellular ECP increased significantly from 0.85 ± 0.007 to 0.92 ± 0.04. The elevated ECP and relative ATP level, together with a significant decrease in the relative AMP level, are all indicators of inhibition of cellular metabolism. These results support the notion that acute exposure of C6 glioma cells to a high concentration of Zn might initially result in a decrease in relative AMP and an inhibition of mitochondrial activity. However, the ultimate toxic action of Zn on the C6 glioma cells appears to be due to a gradual inhibition of energy utilization, leading to cell shrinkage and apoptosis.

Determination of Energy Charge Potential in the C6 Glioma and the HepG-2 Cell Culture

The energy charge potential (ECP) is the ratio among adenosine 5'-triphosphate (ATP), -diphosphate (ADP), and -monophosphate (AMP); it reflects the high-energy-bond content in biological systems. Under normal physiological conditions, the value of ECP is between 0.7 and 1.0. In this study, two different methods were used to extract adenosine nucleotides (ATP, ADP, and AMP) for determination of the ECP in the C6 glioma and the HepG-2 human hepatoma cell lines. In the first method, cells were removed from culture plates by trypsinization before the nucleotides were extracted using perchloric acid. Some of the trypsinized cells were used for a cell count, thereby allowing the calculation of an average nucleotide level per cell. In the second method, perchloric acid was added directly to the culture plate immediately after the medium was removed. This method resulted in an instant termination of any enzymatic process that might degrade the nucleotides and alter their levels in cells. Adenosine nucleotides were determined by high-performance liquid chromatography. The direct extraction method yielded a total adenosine nucleotide (TAN) level more than twofold higher than that obtained following trypsinization. For the C6 glioma cells, the ratio of ATP to ADP to AMP in the TAN pool was 0.70:0.15:0.15, as compared to 0.36:0.27:0.37 when cells were extracted following trypsinization. Similarly, for the HepG-2 cells, the ratio was 0.62:0.15:0.23 following direct extraction, as compared to 0.15:0.28:0.57 following trypsinization. Using the results from the direct extraction method, the value of the cellular ECP was 0.78 ± 0.06 for the C6 glioma cells and 0.70 ± 0.06 for the HepG-2 cells. Compared to the ECP values obtained using the trypsinization method (0.50 ± 0.05 and 0.29 ± 0.12, respectively), the ECP values derived using the direct extraction method were significantly greater in both cell lines and were suggestive of the energy status of normal, healthy cells.

The use of insulin and glucose during resuscitation from hemorrhagic shock increases hepatic ATP.

Hemorrhagic shock produces a marked decrease in hepatic ATP, adenylate energy charge, and total adenosine nucleotides. This is followed by slow recovery to normal levels after resuscitation. Nucleotide metabolites are increased following shock and resuscitation. Previous experimental work has shown that supraphysiologic doses of insulin have salutary effects in animals with hemorrhagic shock and in cardiac patients. It appears that insulin causes increased availability of glucose and energy-producing substrates. This study examined whether resuscitation with glucose and insulin after hemorrhagic shock would alter the changes previously seen to occur in hepatic ATP levels, adenylate energy charge, or nucleotide metabolites. Male Sprague-Dawley rats were bled to a mean arterial blood pressure of 40 mm Hg for 30 min. They were then resuscitated with the shed blood and one of three fluids: (1) lactated Ringer's, (2) lactated Ringer's with 10% glucose, (3) lactated Ringer's with 10% glucose + 6 units/kg regular insulin. Liver biopsies were obtained prior to shock (baseline), after 30 min of shock (shock), and 90 min after resuscitation (90 min). Tissue levels of ATP, ADP, AMP, adenosine, inosine, hypoxanthine, and xanthine were measured. Serum at 90 min was evaluated for potassium, glucose, and tumor necrosis factor alpha (TNF-alpha). The insulin-treated group had significantly increased hepatic ATP and energy charge following resuscitation compared with the other two groups. The insulin group also exhibited significant hypoglycemia. Total adenine nucleotides (ATP, ADP, and AMP) were significantly elevated 90 min postresuscitation in the insulin group. Mean blood pressures throughout the experiment were not significantly different among groups. TNF-alpha was highest in the insulin-treated group, but this was not significant. Resuscitation with insulin and dextrose significantly increased hepatic ATP and adenylate energy charge after hemorrhagic shock in rats. Total nucleotide pool levels were not different between groups, indicating that there was a shift of the equilibrium away from the metabolites toward ATP and ADP in the insulin-treated group. Insulin treatment had no significant effect on blood pressure or TNF-alpha. However, it caused significant hypoglycemia and hypokalemia.

The Alterations of Cellular Metabolism in the Contralateral Testis Following Spermatic Cord Torsion in Rats

Cellular metabolic study was performed on the contralateral testis after torsion to delineate early testicular changes. In 9-week-old Sprague-Dawley rats the left testicle was rotated and fixed to the scrotum for 3, 8 and 24 hours in 3 groups (I, II and III). Each group was divided into 3 subgroups (a, b and c) by the length of detorsion of 0, 4 hours and 14 days. We measured adenosine triphosphate, total adenosine nucleotides and adenylate energy charge of the contralateral testis by high performance liquid chromatography. Compared to controls, no subgroups showed differences in the histological parameters. Also in subgroups Ia, Ib, Ic and IIa none of the metabolic parameters were significantly altered. However, subgroups IIb and IIc, and group III and its subgroups showed significantly decreased levels of all metabolic parameters. Therefore, it appears that unilateral testicular torsion (greater than 8 hours) and detorsion affect the contralateral testicular metabolism without gross histological changes in rats, and these changes seem to be reversible in terms of cellular recoverability because the maximally decreased level of adenylate energy charge among all subgroups is only 13% compared to controls. These findings suggest that some systemic mechanisms rather than an immunological or local inflammatory process is involved in this early period after torsion.

Effects of in Vivo Heart Irradiation on Myocardial Energy Metabolism in Rats

To investigate the effect of in vivo heart irradiation on myocardial energy metabolism, we measured myocardial adenosine nucleotide concentrations and mitochondrial oxygen consumption in left ventricular tissue of rats 0-16 months after local heart irradiation (20 Gy). At 24 h and 2 months no difference in myocardial adenosine nucleotide concentration was apparent between irradiated and control hearts. The total myocardial adenosine nucleotide concentrations in irradiated hearts compared to those of nonirradiated controls tended to be lower from 4 months onward. The rate of oxidative energy production (state 3 respiration) in irradiated hearts was significantly reduced compared with that of age-matched controls from 2 months onward. Moreover, as a result of aging, a time-dependent decrease in the rate of oxidative energy production was observed in both irradiated and control hearts (P < 0.001). The respiratory control index (RCI = oxygen consumption in state 3/oxygen consumption in state 4) in irradiated hearts was not different from the RCI measured in age-matched control animals. During the period of study the RCI diminished significantly with age in both groups (P < 0.005). The number of oxygen atoms used per molecule of ADP phosphorylated (P/O ratio) was not influenced by the irradiation. The P/O ratio for the NAD(+)-linked substrates remained unchanged at a value of about 3 during the period studied. At 6 months after irradiation activities of myocardial enzymes such as lactate dehydrogenase, creatine kinase, citrate synthase, and cytochrome c oxidase were reduced. The reduction in myocardial energy production and the changes in energy supplies provide a mechanism to explain impaired contractility after local heart irradiation.

Phosphagens and glycogen content in skeletal muscle after treadmill training in young and old rats.

The concentration of creatine phosphate (CrP), ATP, ADP, AMP and glycogen were measured in extensor digitorum longus (EDL) and in quadriceps muscles of 3, 6, 24, and 27 months old male Wistar rats groups. Young (3 months) and old (24 months) rats were trained for 12 weeks, 3 days a week, with running exercise session. Each training session was of 2 h. In sedentary groups and for both muscles, CrP, ATP, ADP, and glycogen contents decrease with aging (between 6 and 27 months). In spite of an AMP increase, total adenosine nucleotides (TAN) decrease significantly between 6-27 months (P less than 0.01) from 6.11 to 5.11 (EDL) and from 5.59 to 4.65 (quadriceps) mumol X g-1 wet weight muscle. After 12 weeks of physical training, the mean values of CrP, TAN, and glycogen were improved in both young and old rat groups. Moreover, the ATP/ADP ratios and the energy charge of the adenylate system were unrelated to age, but training decreases significantly the mean value of energy charge in both young and old groups. These results suggest that, as far as energy-rich phosphagen metabolism is concerned young and old muscles show the same pattern response to training.

Low extracellular Ca2+ and enzyme release in the perfused rabbit heart.

Previous studies have shown that the well-oxygenated perfused rabbit heart releases creatine kinase when treated with the calcium antagonist drug verapamil (VER) in a dose-related manner. It is possible that this effect is related to Ca2+ ion deprivation of the sarcolemma. This possibility was explored by perfusing hearts with low Ca2+ (0.5, 0.23, 0.15, and 0 mM) versus a control group (1.27 mM Ca2+) for 60 min. Low Ca2+ perfusion was associated with reduction in the heart rate--left ventricular systolic pressure product and O2 consumption, tendency for the coronary sinus flow to increase, electromechanical dissociation, prolongation of atrioventricular conduction and QT interval, and decrease in myocardial glycogen. Lower total adenosine nucleotides were found only in the 0 mM Ca2+ group. As the Ca2+ concentration was reduced, the hearts lost increasing amounts of creatine kinase, aspartate aminotransferase, and lactate dehydrogenase. These results confirm the importance of Ca2+ ions in contractile and electrical cardiac functions and show that decreased availability of this cation leads to increasing enzyme leakage resembling that seen in VER-treated hearts.

Release of two vasodilators, adenosine and prostacyclin, from isolated rabbit hearts during controlled hypoxia.

The release of two locally formed vasodilators, adenosine and prostacyclin (PGI2), from hearts subjected to different degrees of hypoxia was investigated. Isolated rabbit hearts were perfused according to Langendorff with Tyrode solution, saturated with gas mixtures containing 8-95% O2 and 5% CO2 in N2. Coronary flow rate, O2 extraction and uptake, and cardiac production of lactate, purines and 6-keto-PGF1 alpha (the stable metabolite of PGI2) were determined. During perfusion of the hearts with a solution saturated with 95% O2, release of lactate, 6-keto-PGF1 alpha and purines was very low: lactate was liberated at a rate of about 5 mumol/100 g . min, purine release corresponded to 2% of the total adenosine nucleotide content of the heart per hour and the release of 6-keto-PGF1 alpha was about 150 mumol/100 g . min. During hypoxia there was a graded release of lactate and purines from the heart, as well as a liberation of 6-keto-PGF1 alpha. Mild hypoxia (60% O2 in the gas mixture) elicited a 160% increase in the formation of lactate and a 40% increase in the release of purines. During severe hypoxia (8% O2 in the gas mixture) the release of lactate and purines increased by more than 2000%. In contrast, the release of 6-keto-PGF1 alpha never increased more than 80% at any degree of hypoxia, neither did it correlate to the severity of the hypoxia. From these data we conclude that of the two vasodilating agents formed in the heart, adenosine and prostacyclin, the former is probably more important in the regulation of coronary flow.

Perturbations in the biomass, metabolic activity, and community structure of the estuarine detrital microbiota: Resource partitioning in amphipod grazing

With the development of quantitative methods for estimating the biomass, metabolic activity, nutritional status and community structure of the estuarine detrital microbiota, and the indication of marked effects on the microbiota by amphipod grazing, a detailed examination of the effects of grazing by two sympatric amphipods was conducted. The microbiota, which develop on Teflon squares after a 2-wk exposure in a North Florida estuary, showed, when grazed for 2 wk, significantly increased bacterial biomass (short-branched and cis-vaccenic fatty acids) and microeukaryotes (total polyenoic fatty acids). Grazing in the laboratory at field density further increased the bacterial biomass (muramic acid, wall glucosamine), as well as the total photosynthetic biomass (lipid galactose) if compared to the microbiota grazed in the field. Grazing in the laboratory also depressed the algae estimated as alpha linolenic acid but stimulated the fast growing diatoms (20 carbon polyenoic fatty acids of the alpha linolenic series). The field-grazed microbiota showed metabolic stress indicated by the higher adenosine to adenosine triphosphate (ATP) ratio. Two sympatric amphipods Gammarus mucronatus (Say) and Melita appendiculata (Say) having markedly different mouth parts were shown to partition the detrital microbiota. Melita feed on nonphotosynthetic microeukaryotes (lowered triglyceride glycerol) leaving a larger total biomass (extractable lipid phosphate), photosynthetic biomass (lipid galactose) with greater total metabolic activity estimated as the 4-h rate of phospholipid and sulfolipid synthesis. Bacterial biomass increased (muramic acid, short-branched and cis-vaccenic fatty acids) compared with the detritus grazed by Gammarus. Gammarus feeds less specifically on bacteria and photosynthetic microeukaryotes. Bacteria (muramic acid) and microeukaryotes (the ratio of lipid glycerol to lipid phosphate) were significantly decreased. This feeding provokes an intense metabolic activity from the lowered biomass that is reflected in the significantly higher total adenosine nucleotides, ATP, and adenylate energy charge. The amphipods from the field and laboratory microcosms showed similar adenylate energy charges and neutral lipid glycerol to phospholipid ratios, indicating their nutritional status was similar.

Effect of Light on Biomass and Community Structure of Estuarine Detrital Microbiota

Comparison of estuarine detrital microbiota grown with and without light in the absence of macroscopic grazing showed shifts in the community structure that enabled correlation between various biochemical measures. Analysis of these biochemical measures showed that growth in light induces the smallest increases in procaryotic attributes such as muramic acid; wall glucosamine; lipid phosphate; total extractable adenosine nucleotides; short-branched, cyclopropane, and cis-vaccenic fatty acids; lipid glucose and mannose; the incorporation of acetate into lipid; and the formation of deoxyribonucleic acid from thymidine. Measures of the microfauna such as lipid inositol and the gamma-linolenic series of polyenoic fatty acids also increased minimally in the light-grown microbiota. Measures of sulfo-lipid synthesis, lipid glycerol, total extractable palmitate, 18-carbon polyenoic fatty acids, and total polyenoic fatty acids longer than 20 carbons increased 10- to 15-fold in algae and fungi. Chlorophyll a, lipid galactose, and the 16- and 20- carbon polyenoic fatty acids characteristic of diatoms increased maximally in the light. This increase of diatom measure correlated with the sheets of diatoms detected by scanning electron microscopy.

Studies on the metabolic activities of the bladder tumors. 2. Studies on the energy metabolism of the bladder tumors (author's transl)

Energy metabolism was studied on the specimens of the bladder tumors obtained at operations.Tissue respiration, aerobic and anerobic glycolysis were determined by Warburg's manometric method. Adenosine nucleotides were also analysed by Bucher's method. Inorganic phosphorus contents were determined by Martin-Doty's method. Energy charge was calculated as to the principal adovocated by Atkinson.The results were as follows:1. Anerobic glycolysis was remarkably high in the bladder tumors, although tissue respiration and aerobic glycolysis were activated.2. Warburg's quotient of the bladder tumors was high, which was in good agreement with the results reported for the other malignancies.3. Meyerhof's quotient was found to be high in badder tumors. In addition there existed Pasteur effect. It was assumed that in this malignancy there existed glycolytic inhibition by oxygen or aerobic condition, which had been demonstrated in normal tissues. Therfore, it was considered that there were no great differences between bladder tumors and normal tissues as to the qualities of the respiration and glycolysis.4. Among the adenosine nucleotides, contents of ATP and ADP were elevated in bladder tumors with progressive increase of inorganic phosphorus. Accordingly total adenosine nucleotides were increased as the grades of the tumors progressed.5. It was demonstrated that the bladder tumors had high level of energy charge.6. It was considered that mainly anerobic glycolysis would produce high energy charge, which seemed to be the source of proliferation of this malignancy.