Decrease In High-energy Phosphates Research Articles

Role of nitration in control of phosphorylase and glycogenolysis in mouse skeletal muscle.

Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of the redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to H2O2 had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO-), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half-maximal inhibition at ∼200 µM ONOO-), which was fully reversed by the presence of an ONOO- scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO- resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO- also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ∼60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant N-acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO- inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions.NEW & NOTEWORTHY Here we show that exogenous peroxynitrite results in nitration of phosphorylase as well as inhibition of glycogenolysis in isolated intact mouse skeletal muscle during short-term repeated contractions. However, repeated contractions in the absence of exogenous peroxynitrite do not result in nitration of phosphorylase or affect glycogenolysis, nor does the addition of antioxidants alter glycogenolysis during repeated contractions. Thus phosphorylase is not subject to redox control during repeated contractions.

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Energy status of pig donor organs after ischemia is independent of donor type

BackgroundLiterature is controversial whether organs from living donors have a better graft function than brain dead (BD) and non–heart-beating donor organs. Success of transplantation has been correlated with high-energy phosphate (HEP) contents of the graft. MethodsHEP contents in heart, liver, kidney, and pancreas from living, BD, and donation after cardiac death in a pig model (n=6 per donor type) were evaluated systematically. BD was induced under general anesthesia by inflating a balloon in the epidural space. Ten hours after confirmation, organs were retrieved. Cardiac arrest was induced by 9V direct current. After 10min of ventricular fibrillation without cardiac output, mechanical and medical reanimation was performed for 30min before organ retrieval. In living donors, organs were explanted immediately. Freeze-clamped biopsies were taken before perfusion with Celsior solution (heart) or University of Wisconsin solution (abdominal organs) in BD and living donors or with Histidine-Tryptophan-Ketoglutaric solution (all organs) in non–heart-beating donors, after perfusion, and after cold ischemia (4h for heart, 6h for liver and pancreas, and 12h for kidney). HEPs (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and phosphocreatine), xanthine, and hypoxanthine were measured by high-performance liquid chromatography. Energy charge and adenosine triphosphate-to-adenosine diphosphate ratio were calculated. ResultsAfter ischemia, organs from different donor types showed no difference in energy status. In all organs, a decrease of HEP and an increase in hypoxanthine contents were observed during perfusion and ischemia, irrespective of the donor type. ConclusionOrgans from BD or non–heart-beating donors do not differ from living donor organs in their energy status after average tolerable ischemia.

Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle

Increases in contraction-stimulated glucose transport in fast-twitch rat epitrochlearis muscle are mediated by AMPK- and Ca2+/calmodulin-dependent protein kinase (CAMK)-dependent signaling pathways. However, recent studies provide evidence suggesting that contraction-stimulated glucose transport in slow-twitch skeletal muscle is mediated through an AMPK-independent pathway. The purpose of the present study was to test the hypothesis that contraction-stimulated glucose transport in rat slow-twitch soleus muscle is mediated by an AMPK-independent/Ca2+-dependent pathway. Caffeine, a sarcoplasmic reticulum (SR) Ca2+-releasing agent, at a concentration that does not cause muscle contractions or decreases in high-energy phosphates, led to an approximately 2-fold increase in 2-deoxyglucose (2-DG) uptake in isolated split soleus muscles. This increase in glucose transport was prevented by the SR calcium channel blocker dantrolene and the CAMK inhibitor KN93. Conversely, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, had no effect on 2-DG uptake in isolated split soleus muscles yet resulted in an approximately 2-fold increase in the phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase. The hypoxia-induced increase in 2-DG uptake was prevented by dantrolene and KN93, whereas hypoxia-stimulated phosphorylation of AMPK was unaltered by these agents. Tetanic muscle contractions resulted in an approximately 3.5-fold increase in 2-DG uptake that was prevented by KN93, which did not prevent AMPK phosphorylation. Taken in concert, our results provide evidence that hypoxia- and contraction-stimulated glucose transport is mediated entirely through a Ca2+-dependent mechanism in rat slow-twitch muscle.

Calcium overload and cardiac function.

The changes in cardiac function caused by calcium overload are reviewed. Intracellular Ca(2+) may increase in different structures [e.g. sarcoplasmic reticulum (SR), cytoplasm and mitochondria] to an excessive level which induces electrical and mechanical abnormalities in cardiac tissues. The electrical manifestations of Ca(2+) overload include arrhythmias caused by oscillatory (V(os)) and non-oscillatory (V(ex)) potentials. The mechanical manifestations include a decrease in force of contraction, contracture and aftercontractions. The underlying mechanisms involve a role of Na(+) in electrical abnormalities as a charge carrier in the Na(+)-Ca(2+) exchange and a role of Ca(2+) in mechanical toxicity. Ca(2+) overload may be induced by an increase in [Na(+)](i) through the inhibition of the Na(+)-K(+) pump (e.g. toxic concentrations of digitalis) or by an increase in Ca(2+) load (e.g. catecholamines). The Ca(2+) overload is enhanced by fast rates. Purkinje fibers are more susceptible to Ca(2+) overload than myocardial fibers, possibly because of their greater Na(+) load. If the SR is predominantly Ca(2+) overloaded, V(os) and fast discharge are induced through an oscillatory release of Ca(2+) in diastole from the SR; if the cytoplasm is Ca(2+) overloaded, the non-oscillatory V(ex) tail is induced at negative potentials. The decrease in contractile force by Ca(2+) overload appears to be associated with a decrease in high energy phosphates, since it is enhanced by metabolic inhibitors and reduced by metabolic substrates. The ionic currents I(os) and I(ex) underlie V(os) and V(ex), respectively, both being due to an electrogenic extrusion of Ca(2+) through the Na(+)-Ca(2+) exchange. I(os) is an oscillatory current due to an oscillatory release of Ca(2+) in early diastole from the Ca(2+)-overloaded SR, and I(ex) is a non-oscillatory current due to the extrusion of Ca(2+) from the Ca(2+)-overloaded cytoplasm. I(os) and I(ex) can be present singly or simultaneously. An increase in [Ca(2+)](i) appears to be involved in the short- and long-term compensatory mechanisms that tend to maintain cardiac output in physiological and pathological conditions. Eventually, [Ca(2+)](i) may increase to overload levels and contribute to cardiac failure. Experimental evidence suggests that clinical concentrations of digitalis increase force in Ca(2+)-overloaded cardiac cells by decreasing the inhibition of the Na(+)-K(+) pump by Ca(2+), thereby leading to a reduction in Ca(2+) overload and to an increase in force of contraction.

Preservation of mitochondrial function during ischemia as a possible mechanism for cardioprotection of diltiazem against ischemia/reperfusion injury

A possible mechanism for d- cis-diltiazem (diltiazem)-mediated improvement of the contractile function of ischemic/reperfused hearts was examined. Thirty-five-min ischemia/60-min reperfusion recovered little the left ventricular developed pressure (LVDP) and decreased myocardial high-energy phosphates (HEPs). Ischemia induced an accumulation of tissue Na + content, an increase in cytochrome c in the cytosolic fraction, and a decrease in the oxygen consumption rate (OCR) in perfused hearts. Treatment of the heart with 1 μM diltiazem for the last 3-min of pre-ischemia did not affect the decrease in HEPs during ischemia, whereas that with 3 μM partially attenuated the decrease in ATP, suggesting that 3 μM diltiazem exerted energy-sparing effect. Treatment with 1 μM diltiazem enhanced the post-ischemic recovery of LVDP associated with attenuation of the ischemia-induced accumulation of tissue Na +, increase in cytochrome c in the cytosolic fraction, and decrease in myocardial OCR, and restoration of the myocardial HEPs during reperfusion. Combined treatment with diltiazem and a Na +/H + exchange inhibitor, but not a Na + channel blocker, facilitated the attenuation of Na + accumulation in the ischemic heart and the enhancement of the post-ischemic recovery of LVDP. Sodium lactate, a possible metabolite in ischemic hearts, and sodium chloride increased the Na + concentration in mitochondria, released cytochrome c into incubation medium, and reduced the mitochondrial respiration. Treatment of isolated mitochondria with diltiazem failed to attenuate the sodium lactate- and sodium chloride-induced alterations. These results suggest that the cardioprotection of diltiazem may be exerted via attenuating cytosolic Na + overload through Na + channels in the ischemic heart, leading to preservation of mitochondrial functional ability during ischemia, followed by improvement of post-ischemic energy production and contractile recovery.

Prevention of kainic acid seizures-induced changes in levels of nitric oxide and high-energy phosphates by 7-nitroindazole in rat brain regions

Previous studies using the spin trapping agent N- tert-butyl-α-phenylnitrone (PBN) and the antioxidant vitamin E established the involvement of free radicals in kainic acid (KA)-induced neurotoxicity. In the present study, we examined the effects of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) to establish a possible role of nitric oxide (NO) in the neurotoxicity caused by KA-induced status epilepticus (SE). A single injection of KA (15 mg/kg, s.c.) induced seizures within 40–45 min, progressing to full seizure activity lasting about 3 h. Following microwave (head-focused) irradiation, perchloric acid extracts of rat brain regions (cortex, amygdala, and hippocampus) were analyzed for citrulline (determinant of NO) and high-energy phosphates (HEP) and their metabolites using high-performance liquid chromatograph (HPLC). KA-induced seizures produced a maximum increase in NO (3- to 6-fold) and a decrease in HEP (ATP 45–51% and phosphocreatine 45–58%) 2 h after KA injection in brain regions tested. 7-NI (50 mg/kg, i.p.) when given alone, reduced citrulline/NO levels (10–24%), while repeat administration of 7-NI (60 min apart) reduced NO levels by 32–49%. Neither application of 7-NI produced changes in HEP levels or toxicity. Pretreatment with 7-NI 30 min before KA injection, delayed the onset of seizures by 15–20 min, and significantly prevented an increase in NO and a decrease in HEP. Repeat administration of 7-NI, i.e. 30 min before and 30 min after KA injection, further increased protection by the delayed onset of seizures, attenuating the increase in NO and the decrease in HEP. Neurotoxicity of seizures involves activation of nNOS and of energy consumption in affected neurons. This increased energy consumption, coupled with decreased energy production caused by NO-induced mitochondrial dysfunction, may be a contributing factor to neuronal injury in KA toxicity.

Cardioprotective Effects of Extracts from Psidium guajava L. and Limonium wrightii, Okinawan Medicinal Plants, against Ischemia-Reperfusion Injury in Perfused Rat Hearts

The aim of this study was to determine whether the medicinal herbs growing in Okinawa and possessing a radical-scavenging activity would exert cardioprotective effects against ischemia-reperfusion injury using isolated perfused rat hearts. Effects of the aqueous extracts from Psidium guajava L. and Limonium wrightii at concentrations having an equipotent radical-scavenging activity on myocardial injury produced by global ischemia followed by reperfusion were tested and were further compared with those of quercetin and gallic acid, major antioxidative components of P. guajava L. and L. wrightii, respectively. Both extracts significantly attenuated ischemic contracture during ischemia and improved myocardial dysfunction after reperfusion. Decreases in high-energy phosphates and increases in malondialdehyde in the reperfused hearts were significantly lessened with both plant extracts. Quercetin and gallic acid also exerted similar beneficial effects. These results indicate that P. guajava L. and L. wrightii both have cardioprotective effects against myocardial ischemia-reperfusion injury in isolated rat hearts, primarily through their radical-scavenging actions.

Efficacy of recombinant human Hb by 31P-NMR during isovolemic total exchange transfusion.

The ability of recombinant human Hb (rHb1.1), which is being developed as an oxygen therapeutic, to support metabolism was measured by in vivo 31P-NMR surface coil spectroscopy of the rat abdomen in control animals and in animals subjected to isovolemic exchange transfusion to hematocrit of <3% with human serum albumin or 5 g/dl rHb1.1. No significant changes in metabolite levels were observed in control animals for up to 6 h. The albumin-exchange experiments, however, resulted in a more than eightfold increase in Pi and a 50% drop in phosphocreatine and ATP within 40 min. The tissue pH dropped from 7.4 to 6.8. The decrease in high-energy phosphates obeyed Michaelis-Menten kinetics, with a Michaelis-Menten constant of 3% as the hematocrit at which a 50% drop in high-energy phosphates was observed. Exchange transfusion with rHb1.1 resulted in no significant drop in high-energy phosphates, no rise in Pi, and no change in tissue pH from 7.35 +/- 0.15 for up to 5 h after exchange. By these criteria, rHb1.1 at a plasma Hb concentration of approximately 5 g/dl after total exchange transfusion was able to sustain energy metabolism of gut tissue at levels indistinguishable from control rats with a threefold higher total Hb level in erythrocytes.

Interpretation of blood lactate concentrations in patients with sepsis

Blood lactate concentrations have been used as a marker of tissue hypoperfusion in circulatory shock for over 30 years.1 The rise in lactate concentration correlates with total oxygen debt and with decreases in high-energy phosphates. Under aerobic conditions, 1 mole of glucose is metabolised to pyruvate, which, after conversion to acetyl-coenzyme A, enters the tricarboxylic acid (Krebs) cycle and is oxidised to carbon dioxide with the production of a total of 38 moles of ATP. By contrast, under anaerobic conditions, pyruvate is converted to lactate via the Embden-Meyerhof (glycolytic) pathway, and only 2 moles of ATP are produced per mole of glucose.

Derangement in aerobic and anaerobic energy metabolism in skeletal muscle of critically ill and recovering rats

As part of our research into the mechanisms of protein wasting and muscle weakness during critical illness, we here investigate various aspects of energy metabolism. Intraperitoneal injection of zymosan in rats leads to an acute phase of critical illness followed by a prolonged recovery phase. Previously we observed low activities of mitochondrial enzymes, reduced protein synthesis rates and low concentrations of glutamine in skeletal muscle of zymosan-treated rats. In the present study we investigated (1) whether decreases in high energy phosphates are present in skeletal muscle of these rats and (2) whether an impairment in the glycolytic pathway or the tricarboxylic acid cycle leads to these decreases. Concentrations of creatine phosphate and ATP were decreased in zymosan-treated rats to approx. 85% of pair-fed control values respectively on day 2 and on days 4 and 6 after treatment. Concentrations of tricarboxylic acid (TCA) cycle intermediates were decreased to 80% on day 6 after zymosan treatment. Lactate/pyruvate ratio and concentrations of lactate and glycogen were normal at all sampling times. We conclude that no major changes in concentrations of high energy phosphates and in concentrations of intermediates of TCA cycle, glycolysis and glycogenolysis were present. This indicated that, although the maximal oxidative capacity (mitochondrial content) is decreased, no derangement in energy metabolism seems to be present in skeletal muscle of critically ill and recovering rats.

The role of accumulation of sodium and calcium on contractile failure of the hypoxic/reoxygenated heart.

The present study was undertaken to determine whether myocardial energy or ion levels are related to oxygen-replenishment-induced recovery of cardiac contractile force after hypoxia. Isolated rat hearts were perfused for 3 to 40 min under hypoxic conditions, followed by 45 min of reoxygenation. Hypoxia induced a cessation of cardiac contractile force, a rise in resting tension, a decrease in high energy phosphates, and an increase in lactate. Myocardial ATP, creatine phosphate (CP) and lactate reached steady-state levels after 15, 10 and 5 min of hypoxia, respectively. Hypoxic conditions in the present study also caused an increase in sodium content and a decrease in potassium content, but not changes in calcium content, along with a prolonged hypoxic period. When the hearts were perfused for more than 25 min under hypoxic conditions, no recovery of contractile force was observed following 45-min of reoxygenation. Hypoxic perfusion for more than 25 min induced an accumulation of tissue sodium content approximately 3 fold higher than the pre-hypoxic value at the end of hypoxia, and also induced a marked increase in myocardial calcium content upon reoxygenation. When tissue sodium content accumulated by less than 300% of the pre-hypoxic value, cardiac contractile function was partially reversed by reoxygenation and calcium-overload was not observed. The recovery of post-hypoxic cardiac contractility correlated with tissue sodium content during hypoxia rather than with myocardial high energy phosphate content at the end of hypoxia. These results suggest that accumulation of tissue sodium content in the hypoxic myocardium and calcium content in the reoxygenated myocardium may be indicative of hypoxia/reoxygenation-induced cardiac contractile failure.

Relation of myocardial oxygen consumption and function to high energy phosphate utilization during graded hypoxia and reoxygenation in sheep in vivo.

This study investigates the relation between myocardial oxygen consumption (MVO2), function, and high energy phosphates during severe hypoxia and reoxygenation in sheep in vivo. Graded hypoxia was performed in open-chested sheep to adjust PO2 to values where rapid depletion of energy stores occurred. Highly time-resolved 31P nuclear magnetic resonance spectroscopy enabled monitoring of myocardial phosphates throughout hypoxia and recovery with simultaneous MVO2 measurement. Sheep undergoing graded hypoxia (n = 5) with an arterial PO2 nadir of 13.4 +/- 0.5 mmHg, demonstrated maintained rates of oxygen consumption with large changes in coronary flow as phosphocreatine (PCr) decreased within 4 min to 40 +/- 7% of baseline. ATP utilization rate increased simultaneously 59 +/- 20%. Recovery was accompanied by marked increases in MVO2 from 2.0 +/- 0.5 to 7.2 +/- 1.9 mumol/g per min, while PCr recovery rate was 4.3 +/- 0.6 mumol/g per min. ATP decreased to 75 +/- 6% of baseline during severe hypoxia and did not recover. Sheep (n = 5) which underwent moderate hypoxia (PO2 maintained 25-35 mmHg for 10 min) did not demonstrate change in PCr or ATP. Functional and work assessment (n = 4) revealed that cardiac power increased during the graded hypoxia and was maintained through early reoxygenation. These studies show that (a) MVO2 does not decrease during oxygen deprivation in vivo despite marked and rapid decreases in high energy phosphates; (b) contractile function during hypoxia in vivo does not decrease during periods of PCr depletion and intracellular phosphate accumulation, and this may be related to marked increases in circulating catecholamines during global hypoxia. The measured creatine rephosphorylation rate is 34 +/- 11% of predicted (P < 0.01) calculated from reoxygenation parameters, which indicates that some mitochondrial respiratory uncoupling also occurs during the rephosphorylation period.

The protective potency of two commonly used cardioplegic solutions on cultured endothelial cells exposed to free-oxygen radicals injury

Human umbilical vein endothelial cells were incubated with Bretschneider and St. Thomas II cardioplegic solution followed by a stimulation with cumene hydroperoxide (CHPO), which was used as an oxygen radicals generating agent. A statistically significant decrease of intracellular high energy phosphates (adenosine-5-triphosphate: ATP; creatine phosphate; CP) compared to controls was observed in response to Bretschneider cardioplegia and CHPO. Furthermore, significant rises in prostaglandin I 2 (prostacyclin; PGI 2) production and lipidperoxidation were measured. The authors failed to record such alterations of endothelial cell metabolism for the St. Thomas II cardioplegic solution. They could also demonstrate that the cellular protection against oxygen radicals exerted by the St. Thomas II solution is attributable to procaine. The enhanced cytotoxicity of CHPO observed in presence of the Bretschneider solution was found to be partially caused by its constituent L-histidine, which led to significant decreases of high energy phosphates and increased lipidperoxidation when cells were subsequently treated with CHPO. However, alterations of high energy phosphate content initiated by CHPO and amplified by the Bretschneider solution could not be inhibited by adding procaine. Simultaneous pretreatment of cells with the Bretschneider solution and procaine and stimulation with CHPO resulted in decreases of ATP and CP, as observed using the Bretschneider cardioplegia alone.

Modulation of glutamate-induced intracellular energy failure in neonatal cerebral cortical slices by kynurenic acid, dizocilpine, and NBQX.

The severity and rapidity of acute, glutamate-induced energy failure were compared in live cerebral cortical slices. In each experiment 80 live cerebral cortical slices (350 microns thick) were obtained from neonatal Sprague-Dawley rats, suspended and perfused in a nuclear magnetic resonance (NMR) tube, and studied at 4.7 T with interleaved 31P/1H NMR spectroscopy. NMR spectra, obtained continually, were determined as 5-min averages. Slices were perfused for 60 min with artificial cerebrospinal fluid (ACSF) containing either glutamate alone or glutamate mixed with one of three glutamate-receptor antagonists: kynurenate, dizocilpine (MK-801), and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX). Dose-dependent decreases in high-energy phosphates were studied during glutamate exposure (0.5 to 10 mM), with and without antagonist protection. Energy recovery after glutamate exposures was measured during a 60-min washout with glutamate-free, antagonist-free ACSF. Reversible and irreversible energy failures were characterized by changes in intracellular pH, and by changes in relative concentrations of ATP, phosphocreatine (PCr), and inorganic phosphate. No changes were observed in intracellular levels of N-acetylaspartate and lactate. Some special studies were also done using R-(-)-2-amino-5-phosphonovaleric acid (100 microM) and tetrodotoxin (1 mM) to examine glutamate receptor specificity in this tissue model. Dizocilpine (150 microM) best ameliorated the energy failure caused by 2.0 mM glutamate. With dizocilpine the maximum ATP decrease was only 6 +/- 5%, instead of 35 +/- 7%.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of hypoxia and reoxygenation on human umbilical endothelial cells.

We investigated metabolic changes in human umbilical venous endothelial cells, when these were incubated under hypoxic followed by hyperoxic conditions, thus simulating hypoxia and reoxygenation. The human umbilical venous endothelial cells were incubated with a degassed buffer (oxygen content: 0-0.5%) for either 3 h or 24 h, followed by a 60 min incubation with oxygen-perfused buffer (oxygen content: 100%). Three hours of hypoxia led to a slight decrease in the ATP and creatine phosphate content (-16% +/- 5%), while a pronounced decrease of high energy phosphates (-54% +/- 4%) was observed after 24 h of hypoxia. Reoxygenating the cells after 3 h of hypoxia led to restoration of the content of high energy phosphates, while reoxygenation after 24 h resulted in a strong decrease (-66% +/- 4%). The prostaglandin I2 release during the first 3 h of hypoxia exceeded the release in the following 21 h. In all cases, reoxygenation increased the prostaglandin I2 release. Under normoxic conditions the ratio between oxidised glutathione and reduced glutathione shifted from 1:100 to 1:4.5 after 3 h of hypoxia. The content of lipid peroxidation products was almost unaffected during hypoxia, whereas reoxygenation resulted in a pronounced increase (+380% +/- 60%). The results of this in vitro study suggest that relatively long periods of hypoxia lead to a deficiency of high energy phosphates in the cell. Reoxygenation leads to the formation of oxygen-derived radicals, irrespectively of a prior hypoxia.

Glycolysis is necessary to preserve myocardial Ca2+ homeostasis during beta-adrenergic stimulation.

Although ATP derived from glycolysis represents only a small fraction of total myocardial ATP production, metabolic compartmentation may result in preferential use of glycolytic ATP for certain membrane activities, including pumping of Ca2+ from the cytoplasm. We tested this hypothesis by looking for evidence of Ca2+ overload in normoxic perfused rabbit hearts given iodoacetate (IAA, 50 microM) to block glycolysis and isoproterenol (Iso, 0.05 microM) to stimulate Ca2+ entry. The hearts beat isovolumically and were perfused with 16 mM glucose and 5 or 10 mM pyruvate (to preserve oxidative metabolism) in a superconducting magnet for 31P-nuclear magnetic resonance (NMR) measurements of high energy phosphates or 19F-NMR measurements of intracellular free Ca2+ concentration ([Ca2+]i). IAA by itself had no effect on left ventricular (LV) developed pressure, end-diastolic pressure, pressure-rate product, or tissue high-energy phosphates. During exposure to Iso, mean LV end-diastolic pressure increased from 10.7 to 49.3 mmHg in hearts pretreated with IAA (n = 7) but did not change in control hearts (n = 7). During Iso, there were substantial reductions in developed pressure, ATP, and phosphocreatine in IAA-treated hearts but not in control hearts. After exposure to IAA and Iso, a doubling of diastolic [Ca2+]i was observed with 19F-NMR. In IAA-treated hearts, reduction of perfusate Ca2+ concentration from 2.5 to 0.6 mM during Iso exposure (n = 6) prevented the mechanical dysfunction and decrease in high-energy phosphates. These findings suggest that glycolysis is necessary to preserve myocardial Ca2+ homeostasis during beta-adrenergic stimulation.

Correlation of contractile dysfunction and abnormal tissue energy metabolism during hypoperfusion with norepinephrine in isolated rat hearts: Differences between normal and diabetic hearts

The relationship of myocardial high-energy phosphate depletion and lactate accumulation with contractile dysfunctions was investigated in streptozotocin-diabetic (DM) and normal rat hearts. The isolated hearts were perfused with 10 −6 m norepinephrine (NE) at various low-flow rates (0.4–6 ml/min/g heart wt) for 1 h. Left ventricular pressure (LVP) and contractile force (CF) were monitored, through a water-filled balloon in LV and through a hook attached to the apex, respectively. In DM hearts resting CF (diastolic tension) increased, when the perfusion flow rate was reduced below 6 ml and reached a maximum at a flow rate of less than 3 ml. The large increase in LV stiffness correlated with an elevation in diastolic LVP. In normal hearts these parameters were elevated at a flow rate below 1 ml. A flow-dependent decrease in developed CF was more prominent in DM than in normal hearts, while developed LVP and perfusion pressure were slightly higher in DM hearts with a marked increase in the LV stiffness. A flow-dependent decrease in high-energy phosphates and increases in inorganic phosphate and lactate were more prominent in the inner than in the outer layer of LV free wall in both groups. The change of ATP in the inner layer was greater, while increases of lactate in both layers were smaller in DM hearts. Changes in mechanical parameters correlated well with the ATP decrease and lactate increase in the inner layer in both groups. The correlation curves, however, were not coincidental: at the same low ATP and high lactate level, the LV stiffness was higher in DM hearts. Results indicate that DM hearts are more susceptible to flow-reduction with NE and depletion of total ATP in their tissue, and easily suffer from increased LV stiffness. This cannot be explained by the rate of decrease in total ATP and lactate accumulation alone.

O-003 - Decrease in high-energy phosphates in the cerebral artery during chronic spasm.

O-003 - Decrease in high-energy phosphates in the cerebral artery during chronic spasm.