High-energy Phosphate Levels Research Articles

Lactate utilization as an energy substrate in ischemic preconditioned rat brain slices

We examined the utilization of lactate as an energy substrate in ischemic preconditioned slices obtained from the rat brain left hemisphere, of which the contralateral middle cerebral artery was occluded 48 h before the slice preparation. The levels of high-energy phosphates in the brain slices were measured using 31P NMR with a time resolution of 4 min at 25 °C. When iodoacetic acid-pretreated brain slices were further treated with fluorocitrate, a glial toxin, for 2 h (neuron-rich slices), the recovery of the phosphocreatine (PCr) level in artificial cerebrospinal fluid (ACSF) containing lactate after high-K + stimulation was completely abolished in intact slices, whereas the PCr level in ischemic preconditioned slices well recovered in otherwise similar conditions. These results indicated that neurons, when preconditioned with ischemia, acquire the ability to utilize lactate as an energy substrate. In parallel experiments, we recorded population excitatory postsynaptic potentials and spikes from granule cells in hippocampal slices. Population spikes of intact slices in ACSF containing lactate were completely abolished in 30 min, but those of the ischemic preconditioned slices were maintained well over 50%. These results show that ischemic preconditioning may induce certain systematic changes in neurons, such as the expression of lactate transporters and/or the activation of lactate dehydrogenase.

Absolute concentrations of high-energy phosphate metabolites in normal, hypertrophied, and failing human myocardium measured noninvasively with 31P-SLOOP magnetic resonance spectroscopy

ObjectivesThe purpose of the present study was to measure absolute concentrations of phosphocreatine (PCr) and adenosine triphosphate (ATP) in normal, hypertrophied, and failing human heart. BackgroundConflicting evidence exists on the extent of changes of high-energy phosphate metabolites in hypertrophied and failing human heart. Previous reports using phosphorus-31 magnetic resonance spectroscopy (31P-MRS) have quantified metabolites in relative terms only. However, this analysis cannot detect simultaneous reductions. MethodsFour groups of subjects (n = 10 each), were studied: volunteers and patients with hypertensive heart disease (HHD), aortic stenosis, and dilated cardiomyopathy (DCM). Left ventricular (LV) function and mass were measured by cine magnetic resonance imaging. Absolute and relative concentrations of PCr and ATP were determined by 31P-MRS with spatial localization with optimum pointspread function. ResultsLeft ventricular ejection fraction remained normal in HHD and aortic stenosis, but was severely reduced to 18% in DCM; LV mass was increased by 55%, 79%, and 68% respectively. In volunteers, PCr and ATP concentrations were 8.82 ± 1.30 mmol/kg wet weight and 5.69 ± 1.02 mmol/kg wet weight, and the PCr/ATP ratio was 1.59 ± 0.33. High-energy phosphate levels were unaltered in HHD. In aortic stenosis, PCr was decreased by 28%, whereas ATP remained constant. In DCM, PCr was reduced by 51%, ATP by 35%, and reduction of the PCr/ATP ratio by 25% was of borderline significance (p = 0.06). Significant correlations were observed among energetic and functional variables, with the closest relations for PCr. ConclusionsIn human heart failure due to DCM, both PCr and ATP are significantly reduced. Ratios of PCr to ATP underestimate changes of high-energy phosphate levels.

Is the UW solution adequate as an initial flush solution for heart procurement? a comparative study of celsior and UW solutions

The University of Wisconsin (UW) solution has been widely utilized in the process of organ preservation. Its high potassium content and high viscosity however, have been reported to cause vasoconstriction or endothelial damage during heart procurement. We previously reported that the Celsior solution with its high buffer capacity, low potassium content, and low viscosity was equivalent to or superior to the UW solution for cardiac preservation. In this study, myocardial temperature during the washout of coronary vascular beds and myocardial energy metabolism soon after the excision of hearts were evaluated by focusing on the initial flush effect of both solutions. Adult mongrel dogs weighing 9.5 to 13.5 kg were used. Following cardiac arrest using cardioplegia, the heart was cooled with ice slush and coronary vascular beds were washed out with a 4°C UW solution (UW group; n = 5) or Celsior solution (Celsior group; n = 5) at a pressure of 80 cm H2O. During the period of initial washout using 200 ml of solution, both the myocardial temperature and the time required to reach myocardial temperature of 15°C, were noted. The time required to complete the initial washout was also measured. Phosphocreatine (PCr), ß-adenosine triphosphate (ATP) and inorganic phosphate (Pi) levels were measured by using 31P-nuclear magnetic resonance spectroscopy soon after the excision of hearts. PCr/Pi and ß-ATP/Pi values were identified as indicators of myocardial high-energy phosphate levels. The Celsior group tended to require a shorter time to reach a myocardial temperature of 15°C compared with the UW group. The total initial washout time was significantly shorter in the Celsior group than in the UW group. PCr/Pi and ß-ATP/Pi values were significantly higher in the Celsior group than in the UW group. The Celsior solution appears to have significant advantages when used as an initial flush solution compared with the UW solution in heart procurement.

Ischemic Preconditioning Prior to Aortic Cross-Clamping Protects High-Energy Phosphate Levels, Glucose Uptake, and Myocyte Contractility

Background. We hypothesized that indices of myocyte contractility and metabolism could be preserved with ischemic preconditioning in a model of reversible ischemia similar to that occurring during routine cardiac surgery. Regional measures of metabolism and function have not been studied in conjunction with individual myocyte function during postischemic recovery of preconditioned myocardium.Patients and methods. In 16 dogs, myocardium supplied by the left anterior descending artery (LAD) was preconditioned with intermittent LAD ischemia and reperfusion. Following preconditioning, the heart was made globally ischemic for 20 min at normothermia by aortic cross-clamping while on cardiopulmonary bypass. In 10 animals, serial measurements of LAD and remote region adenosine triphosphate (ATP) levels, glucose uptake, and wall thickening were obtained with full-thickness drill biopsies, positron emission tomography (PET), and 2-D echocardiography, respectively. In the remaining 6 animals, cardiac myocytes were isolated after 1 h of reperfusion for measurement of myocyte contractility and intracellular calcium transients.Results. ATP levels were higher in the preconditioned LAD region than in the remote region at end of ischemia (3.17 ± 0.33 nmol/mg vs 2.59 ± 0.30 nmol/mg, P = 0.006). Similarly, preconditioned region glucose uptake was 40% higher than remote region glucose uptake at 2 days postischemia (0.35 ± 0.06 μmol/min/g vs 0.25 ± 0.05 μmol/min/g, P = 0.019). There were no differences in regional wall thickening as measured by 2-D echo either immediately following ischemia or at 2 days. Individual myocyte contractile response to increasing concentrations of extracellular calcium was preserved in cells from preconditioned myocardium, but it was severely depressed in remote region myocytes.Conclusions. We conclude that regional ischemic preconditioning prior to prolonged ischemia protects myocardial glucose uptake and myocyte contractile function. The beneficial effects on glucose metabolism suggest that preconditioning may have sustained protective effects on cell metabolism.

Induction of peritoneal sepsis increases the susceptibility of isolated hearts to a calcium paradox-mediated injury.

The present study was designed to test the hypothesis that induction of chronic peritoneal sepsis in rats would produce a more severe calcium paradox-mediated myocardial injury in isolated heart preparation than is seen in normal hearts, and that this would be inhibited by sucrose as in normal hearts. Male Sprague-Dawley rats were made septic using 200 mg of cecal material (obtained from a donor rat) suspended in 5 mL of 5% dextrose in sterile water D5 W/kg. In septic animals, the cecal material was injected in the peritoneum, while sham-septic animals received only D5 W/kg (5 mL/kg). A third group consisting of normal rats (no surgery) group was also included. Hearts were harvested from all three groups and were subjected to a calcium paradox-mediated injury in an isolated heart preparation. Hearts were perfused with Krebs-Henseleit (KH) medium and were allowed to stabilize, followed by a perfusion with Ca2+-free KH for 10 min. After this 10-min Ca2+-free KH perfusion, rats were reperfused with KH medium for 60 min. Ca2+-free KH medium was used in control experiments, while sucrose experiments were conducted with the same medium except that 150 mM sucrose replaced 75 mM NaCl. A marked decrease in ATP and phosphocreatine occurred during Ca2+ reperfusion in all hearts in absence of sucrose. In the presence of the disaccharide, no change in high-energy phosphate (HEP) levels was observed in normal hearts, while lower ATP concentrations were seen in sham and septic hearts. Thus, sucrose did not inhibit cellular injury in sham and septic hearts as it did in normal hearts, and this might be due to a smaller HEP availability. Control studies with normal, sham, and septic hearts exhibited cessation of contractions in the absence of Ca2+, and appearance of large amounts of cytosolic protein in the effluent perfusate during Ca2+ reperfusion. With normal hearts, perfusion with sucrose caused a 96% inhibition of the total creatine kinase (CK) release observed in control experiments. With sham hearts, 32% of CK release was inhibited by sucrose, while 68% of the CK release was attributed to stress associated with surgery performed in the sham-septic group. In septic hearts, only 8% of the CK release was inhibited by sucrose, suggesting that more severe myocardial injury occurs when septic hearts are subjected to a calcium paradox as compared to other groups. It is evident that sucrose can inhibit a small fraction of the CK release from septic hearts during the calcium paradox as compared to the large CK loss associated with sham sepsis. We have concluded that induction of sepsis made the heart more susceptible to a calcium paradox-mediated myocardial injury.

Effect of dexamethasone on mitochondrial maturation in the fetal rat brain

OBJECTIVE: The objective of the present study was to explore whether prenatal dexamethasone treatment influences mitochondrial maturation in the fetal rat brain. STUDY DESIGN: Mitochondrial respiration was measured polarographically with homogenates of fetal cerebral cortical tissues on day 16 (with saline solution, n = 8; with dexamethasone, n = 8), day 18 (with saline solution, n = 8; with dexamethasone, n = 8), and day 20 (with saline solution, n = 8; with dexamethasone,n = 8) of gestation. Four doses of dexamethasone (0.1 mg · kg) or vehicle (saline solution) were given, withan interval of 12 hours, until 12 hours before each measurement. RESULTS: In the vehicle-treated animals, mitochondrial respiratory activity was increased significantly after day 18 of gestation. Dexamethasone-treated animals showed a significant increase in mitochondrial activity at day 16 of gestation compared with vehicle-treated animals. CONCLUSION: The results indicate that prenatal dexamethasone treatment contributes to the precocious maturation of mitochondrial activity in the fetal rat brain. Because acceleration in cerebral mitochondrial activities is required immediately after birth to maintain high-energy phosphate levels, the precocious maturation may be crucial for the successful outcome of the preterm infant. (Am J Obstet Gynecol 2002;186:574-8.)

Endothelial cell compatibility of azithromycin and erythromycin.

Phlebitis is a severe local adverse event related to the use of parenteral macrolides. In order to evaluate the effect of azithromycin and erythromycin on human venous endothelial cells, we set up an in vitro model. The intracellular levels of purine nucleotides, as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and guanosine 5'-triphosphate (GTP), were measured by means of high-performance liquid chromatography. Incubation of cells with 2 mg/mL azithromycin and erythromycin resulted in a rapid decline of intracellular ATP from 12.5 +/- 0.9 nmol/million cells to 4.1 +/- 0.3 and 2.6 +/- 0.4 nmol/million cells, respectively, after 60 min. In addition, ADP was extensively depleted from 2.1 +/- 0.17 nmol/million cells to 0.8 +/- 0.09 and 0.8 +/- 0.13 nmol/million cells after 60 min. After exposure of 0.5 mg/mL azithromycin and erythromycin, no significant decline of intracellular high-energy phosphate levels occurred after 20 and 60 min. Based on these results, solutions of azithromycin and erythromycin may not be well tolerated and may cause local adverse reactions even if diluted according to the manufacturer's recommendation.

Functional inactivation by interleukin-1beta of glyceraldehyde-3-phosphate dehydrogenase in insulin-secreting cells.

It is well established that long-term exposure of isolated beta cells to cytokines [e.g., IL-1beta] results in increased expression of inducible nitric oxide synthase and subsequent release of nitric oxide, which in turn, has been shown to mediate a wide array of effects, including alterations in cellular high-energy metabolism. In this context, several extant studies have demonstrated significant reduction in adenine and guanine nucleotide triphosphate levels in beta cells exposed to IL-1beta. Herein, we examined the functional status of glyceraldehyde-3-phosphate dehydrogenase [GAPDH] in insulin-secreting cells exposed to IL-1beta, since it represents the first enzyme in the glycolytic pathway that is involved in the generation of ATP. GAPDH was assayed spectrophotometrically in the cytosolic fraction derived from control and IL-1beta -treated [300 pM for 24 hrs] insulin-secreting cell lines [HIT-T15 and RINm5F]. IL-treatment resulted in marked attenuation of GAPDH activity in HIT and RIN cells; such a reduction in this activity was not due to inhibition of its expression by IL-1. Instead, we observed that incubation of HIT and RIN lysates with peroxynitrite, a reactive intermediate of nitric oxide with superoxide anion, resulted in significant reduction in the GAPDH activity. These results identify a GAPDH as one of the biochemical loci for the effects of IL-derived peroxynitrite in the islet beta cell. The previously reported reduction in high-energy phosphate levels in an IL-treated beta cell may, in part, be due to inhibition of GAPDH activity, and subsequent reduction in the glycolytic efficiency of the beta cell.

Role of endogenous endothelin on coronary reflow after cardioplegic arrest

Objective: Endothelin plays a role in the regulation of basal coronary tone. We hypothesized that low coronary reflow and reduced cardiac function after prolonged ischemia may be due to increased release of endogenous endothelin. Methods: Using an isolated perfused rat heart, we examined the effect of the addition of various endothelin antagonists during reperfusion after 4 hours of cardioplegic arrest at 4°C. Hearts were freeze-clamped at the end of reperfusion for analysis of high-energy phosphate levels. Results are expressed as the percentages of preischemic values. Results: The addition of bosentan or Ro61-0612 (nonselective endothelin antagonists) resulted in a significant increase in the recovery of coronary flow after 30 minutes of reperfusion (100.9% vs 85.3% [P =.03] and 122.4% vs 83.7% [P <.001], respectively, versus controls). The addition of PD155080 (endothelin A antagonist) had a similar effect (129.5% vs 91.4%, P =.008). BQ788 (endothelin B antagonist) and phosphoramidon (endothelin-converting enzyme inhibitor) had no effect. Myocardial adenosine triphosphate levels were significantly (12.1%) higher after reperfusion with Ro61-0612 (18.1 ± 0.4 μmol/g vs 16.2 ± 0.5 μmol/g, P =.01). There was no difference in the recovery of cardiac mechanical function with any of the antagonists studied. Conclusion: These results suggest that endogenous endothelin plays a role in low coronary reflow after prolonged cardioplegic arrest but does not impair recovery of myocardial function.

Effect of ethanol on the metabolism of primary astrocytes studied by 13C‐ and 31P‐NMR spectroscopy

Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line (31)P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol.

Opioid preconditioning: myocardial function and energy metabolism

Background. Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with d-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. Methods. After pretreatment with d-Ala2-Leu5-enkephalin, morphine sulfate, or saline (n = 6 each), swine hearts were excised and stored for 75 minutes at 4°C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. Results. Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were observed in hearts pretreated with d-Ala2-Leu5-enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-I) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioid-pretreated hearts ( p < 0.01) at 75 minutes of reperfusion. Conclusions. d-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects.

Effects of augmented delivery of pyruvate on myocardial high-energy phosphate metabolism at high workstate.

This study was performed to determine whether the fall in myocardial high-energy phosphates (HEP) that occurs during high workstates can be ascribed to either inadequate glycolytic pyruvate generation and conversion to acyl-CoA or limitation of long-chain fatty acid transport into the mitochondria. This was tested by using infusions of either pyruvate or butyrate in anesthetized dogs. Pyruvate was used because it bypasses the glycolytic sequence of reactions, activates pyruvate dehydrogenase, and increases mitochondrial NADH concentration ([NADH(m)]) in isolated myocardium, whereas butyrate enters the mitochondria without need for transport by the rate-limiting, palmitoyl-carnitine transporter. Increasing blood pyruvate from 0.16 +/- 0.016 mM to >3 mM did not alter baseline HEP levels determined with (31)P nuclear magnetic resonance, but caused an increase in the rate-pressure product and a modest increase in myocardial oxygen consumption (MVO(2)). Infusion of dobutamine + dopamine (each 20 microg x kg(-1) x min(-1) iv) increased MVO(2) and caused decreases of myocardial phosphocreatine (PCr)/ATP. Pyruvate partially reversed the decrease of HEP levels produced by catecholamine stimulation, whereas butyrate had no effect. Neither pyruvate nor butyrate caused an increase of MVO(2) during catecholamine infusion. Deoxymyoglobin was not detected by (1)H magnetic resonance spectroscopyy in any group. The data demonstrate that carbon substrate availability to the mitochondria is not the only cause of the reduction of PCr/ATP that occurs at high workstates. Supplemental pyruvate (but not butyrate) attenuated the reduction of PCr/ATP during the high workstates; this may have resulted from direct effects on intermediary metabolism or from other effects such as the free radical scavenging activity of pyruvate.

Mitochondrial ATPase and high-energy phosphates in failing hearts.

This study examined high-energy phosphates (HEP) and mitochondrial ATPase protein expression in hearts in which myocardial infarction resulted in either compensated left ventricular remodeling (LVR) or congestive heart failure (CHF). The response of HEP (measured via (31)P magnetic resonance spectroscopy) to a modest increase in the cardiac work state produced by dobutamine-dopamine infusion and pacing (if needed) was examined in 17 pigs after left circumflex coronary artery ligation (9 with LVR and 8 with CHF) and compared with 7 normal pigs. In hearts with LVR, the baseline phosphocreatine (PCr)-to-ATP ratio decreased, and calculated ADP increased; these changes were most severe in hearts with CHF. HEP levels did not change in normal or LVR hearts during dobutamine-dopamine infusion. However, in hearts with CHF, the PCr-to-ATP ratio decreased further, and free ADP increased. The mitochondrial protein levels of the F(0)F(1)-ATPase subunits were normal in hearts with compensated LVR. However, in failing hearts, the alpha-subunit decreased by 36%, the beta-subunit decreased by 16%, the oligomycin sensitivity-conferring protein subunit decreased by 40%, and the initiation factor 1 subunit decreased by 41%. Thus in failing hearts, reductions in mitochondrial F(0)F(1)-ATPase protein expression are associated with increased myocardial free ADP.

Sarpogrelate diminishes changes in energy stores and ultrastructure of the ischemic-reperfused rat heart

Although the involvement of serotonin in exacerbating vascular abnormalities in ischemic heart disease has been established, its role in mediating changes in cardiac function due to ischemia reperfusion (IR) is poorly understood. The aim of this study was to investigate the effect of a serotonin blocker, sarpogrelate (5-HT2A antagonist), in preventing cardiac injury due to IR. Isolated rat hearts were subjected to 30 min of global ischemia followed by 1 h of reperfusion. Sarpogrelate (50 nM-0.9 microM) was infused 10 min before ischemia as well as during the reperfusion period. The IR-induced changes in left ventricular developed pressure, left ventricular end diastolic pressure, rate of pressure development, and rate of pressure decay were attenuated (P < 0.05) with sarpogrelate treatment. Sarpogrelate also decreased the ultrastructural damage and improved the high energy phosphate level in the IR hearts (P < 0.05). This study provides evidence for the attenuation of IR-induced cardiac injury by 5-HT2A receptor blockade and supports the view that serotonin may contribute to the deleterious effects of IR in the heart.

Optimal alveolar oxygen concentration for cold storage of the lung.

Ischemia of the lung is different from that of solid organs because the lung contains gas in the alveoli. However, the optimal gas composition in the alveoli during cold storage remains uncertain. We investigated the relationship between the alveolar oxygen concentration and reperfusion injury. The lungs inflated with 0% O2, 5% O2, room air, 50% O2, or 100% O2 were reperfused after 8 hR storage at 4 degrees C and pulmonary functions were measured for 120 min using an ex vivo rat lung model. The levels of high-energy phosphate and lipid peroxidation of the lung were analyzed after a PA flush, preservation, and reperfusion. Additionally, respiration of the mitochondria in the lungs was measured after preservation. The pulmonary functions were significantly superior in the 5% O2 group than those in the 0% O2, 50% O2, and 100% O2 groups. Pulmonary edema developed in the 0% O2, 50% O2, and 100% O2 groups, but not in the 5% O2 group. After preservation, the energy level in the lungs decreased only in the 0% O2 group. Although lipid peroxidation of the lungs did not increase in any group after preservation, significant increases were observed in the room air, 50% O2 and 100% O2 groups after reperfusion. State 3 and 4 ratios of the mitochondrial respiration significantly decreased in the lungs of the room air, 50% O2 and 100% O2 groups. Although the cold-preserved lungs require oxygen, hyperoxygenation induced mitochondrial dysfunction and increased lipid peroxidation and led to deleterious lung function after reperfusion. Therefore, hypoxic conditions that can maintain the energy level of the lung during cold storage would be optimal.

Phosphocreatine and ATP content in human single muscle fibres before and after maximum dynamic exercise.

The recovery of high-energy phosphate levels in single human skeletal muscle fibres following short-term maximal (all-out) exercise was investigated. Three male volunteers exercised maximally for 25 s on an isokinetic cycling ergometer. Muscle biopsy samples from the vastus lateralis were collected at rest, immediately post-exercise and at 1.5 min of recovery. The subjects also performed a second exercise bout 1.5 min after the first, on a separate occasion. Single muscle fibres were dissected, characterized and assigned to one of four groups according to their myosin heavy chain (MyHC) isoform content; namely, type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fibres were analysed for adenosine 5'-triphosphate (ATP), inosine-5'-monophosphate (IMP), phosphocreatine (PCr) and creatine (Cr) levels. Type I fibres had a lower Cr content than type II fibres (P<0.01). Within type II fibres resting [PCr] increased with increasing MyHC IIX isoform content (r=0.59, P<0.01). Post-exercise [PCr] was very low in all fibre groups (P<0.01 versus rest) while great reductions in ATP were also observed (P<0.01 versus rest), especially in the type II fibre groups. [PCr] at 1.5 min of recovery was still lower compared to rest for all fibre groups (P<0.01) especially in the IIAx and IIXa fibres.

Metabolism changes in single human fibres during brief maximal exercise.

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 +/- 1 and 18 +/- 4 mmol (kg dry mass)(-1), respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP].

Depletion of Energy Metabolites Following Acetylcholinesterase Inhibitor-Induced Status Epilepticus: Protection by Antioxidants

Status epilepticus (SE)-induced neuronal injury may involve excitotoxicity, energy impairment and increased generation of reactive oxygen species (ROS). Potential treatment therefore should consider agents that protect mitochondrial function and ROS scavengers. In the present study, we examined whether the spin trapping agent N- tert-butyl-α-phenylnitrone (PBN) and the antioxidant vitamin E (DL-α-tocopherol) protect levels of high-energy phosphates during SE. In rats, SE was induced by either of two inhibitors of acetylcholinesterase (AChE), the organophosphate diisopropylphosphorofluoridate (DFP, 1.25 mg/kg, sc)- or the carbamate carbofuran (1.25 mg/kg, sc). Rats were sacrificed 1 h or 3 days after onset of seizures by head-focused microwave (power, 10 kW; duration 1.7 s) and levels of the energy-rich phosphates adenosine triphosphate (ATP) and phosphocreatine (PCr) and their metabolites adenosine diphosphate (ADP) and adenosine monophosphate (AMP), and creatine (Cr), respectively, were determined in the cortex, amygdala and hippocampus. Within 1 h of seizure activity, marked declines were seen in ATP (34–60%) and PCr (25–52%). Total adenine nucleotides (TAN=ATP+ADP+AMP) and total creatine compounds (TCC=PCr+Cr) were also reduced (TAN 38–60% and TCC 25–47%). No changes in ATP/AMP ratio were seen. Three days after the onset of seizures, recovery of ATP and PCr was significant in the amygdala and hippocampus, but not in the cortex. Pretreatment of rats with PBN (200 mg/kg, ip, in a single dose), 30 min before DFP or carbofuran administration, prevented induced seizures and partially prevented depletion of high-energy phosphates. Pretreatment with the natural antioxidant vitamin E (100 mg/kg, ip/day for 3 days), partially prevented loss of high energy phosphates without affecting seizures. In controls, citrulline, a product of nitric oxide synthesis, was found to be highest in the amygdala, followed by hippocampus, and lowest in the cortex. DFP- or carbofuran-induced seizures caused elevation of citrulline levels seven- to eight-fold in the cortex and three- to four-fold in the amygdala and hippocampus. These results suggest a close relationship between SE, excitotoxicity and energy metabolism. The involvement of oxidative stress is supported by the findings that DFP and carbofuran trigger an excessive nitric oxide (NO) production in the seizure relevant regions of the brain.

Effect of Graded Hypoxia on Cerebral Cortical Genomic DNA Fragmentation in Newborn Piglets

Previous studies have shown that hypoxia is associated with modification of the cerebral cortical nuclear membrane, leading to increased intranuclear calcium. The increased intranuclear calcium activates calcium-dependent endonucleases, resulting in DNA fragmentation. The present study tests the hypothesis that the fragmentation of neuronal genomic DNA increases with an increase in the degree of cerebral tissue hypoxia. Sixteen newborn piglets were anesthetized, ventilated and divided into normoxic and hypoxic groups with varying degrees of hypoxia. Cerebral hypoxia was documented biochemically by measuring tissue levels of ATP and phosphocreatine. Isolation of cerebral cortical neuronal nuclei and DNA and their purity was confirmed by standard techniques. DNA samples were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. In the hypoxic samples, multiple low-molecular-weight DNA fragments were present as a smear pattern from 200 to 2,000 base pairs. Levels of high-energy phosphates were compared to the area of each smear for each animal to correlate the degree of hypoxia with the degree of DNA fragmentation. DNA fragmentation increased when high-energy phosphate levels decreased. We conclude that there is a critical threshold value of oxidative metabolism beyond which there are progressive changes in the cortical neuronal cells, leading to DNA fragmentation.

High-energy phosphate metabolism and creatine kinase in failing hearts: a new porcine model.

This study aimed to create a pig model of heart failure secondary to severe aortic stenosis and to examine the relationship between the alterations in myocardial high-energy phosphate (HEP) metabolism and protein expression of creatine kinase (CK) isoforms. Sixteen pigs with left ventricular hypertrophy (LVH) secondary to ascending aortic banding and 10 normal pigs (N) were studied. Myocardial protein levels of CK isoforms (Western blot), HEP levels, and CK kinetics ((31)P MR spectroscopy) were measured under basal conditions. Nine of the 16 animals with LVH developed congestive heart failure (CHF), as evidenced by ascites (100 to 2000 mL). LV weight/body weight ratio (g/kg) was 2.18+/-0.15 in N hearts, 3.04+/-0.14 in hearts with LVH (P<0.01), and 4.23+/-0.36 in hearts with CHF (P<0.01 versus LVH). Right ventricle weight/body weight ratio and LV end-diastolic pressure were significantly higher in hearts with CHF (each P<0.01 versus N or LVH). Myocardial phosphocreatine/ATP ratios and the CK forward flux rates were decreased in LVH hearts, most severely in hearts with CHF. CK-M/beta-actin ratios were 2.21+/-12 (N), 1.69+/-0.15 (LVH), and 1.39+/-0.27 (CHF, P<0.05 versus N). CK-mitochondria (CK-Mt)/beta-actin ratios were 1.40+/-0.09 (N), 1.24+/-0.09 (LVH), and 1.02+/-0.08 (CHF, P<0.05 versus N or LVH). The severity of the reduction of CK flux rate was linearly related to the severity of the decrease of CK-Mt/beta-actin (r=0.68, P<0.01). In this new model of heart failure/hypertrophy, the abnormal myocardial HEP metabolism is related to the decreased CK-Mt protein level, which in turn is related to the severity of the hypertrophy.