Exercise metabolism in Duchenne muscular dystrophy: a biochemical and [31P]-nuclear magnetic resonance study of mdx mice

Intracellular pH, ratios of phosphocreatine (PCr) to ATP and PCr to inorganic phosphate (Pi) as well as isometric tension were measured during 1 Hz sciatic nerve stimulation and during recovery in the calf muscles of mdx (a model of Duchenne muscular dystrophy) and control mice. Tension did not decline significantly in either strain. The ratio of PCr/(PCr + Pi) was significantly reduced in mdx as against control muscle during exercise and recovery, but the ratio of PCr/ATP and the half-time for PCr recovery were similar in both strains. A reduction in the maximal activities of succinate dehydrogenase and succinate-cytochrome c reductase suggests that mitochondrial metabolism may be impaired. The similarity in PCr recovery times suggests that the muscle has adapted, making any impairment of oxidative metabolism negligible in the intact system. The rate of pH recovery is prolonged in mdx muscle and provides strong evidence for a decline in the capacity of dystrophic muscle to extrude proton equivalents. These data are compared with a previous study which used 10 Hz stimulation and also observed a slow pH recovery. The slow pH recovery could be explained by an elevation in intracellular sodium.     

A metabolism study of human masseter muscle by 31P magnetic resonance spectroscopy during long periods of exercise and recovery

The metabolism of the human masseter muscle was investigated using phosphorus (31p) magnetic resonance spectroscopy (MRS) during long periods of exercise and recovery. Eleven subjects aged 19 to 28 yr were examined by 31p MRS during four consecutive periods of 13 min each: rest, exercise, recovery 1 and 2. For each subject, a biting force equal to 20% of maximum voluntary biting force was applied and controlled during the exercise period to produce maximum fatigue. 31p MR spectra were localized from a 24 cm3 volume of interest using an image selected in vivo spectroscopy (ISIS) sequence and a 6 cm diameter surface coil placed on the left masseter. Compared to the resting level, the phosphocreatine (PCr) content decreased by 26% during exercise, while the inorganic phosphate (Pi) concentration increased by 65%. During the two recovery periods, the Pi content remained decreased compared with the resting level by 36% and 30%, respectively. The Pi/PCr ratio was increased from 0.30+/-0.04 at rest to 0.63+/-0.13 during exercise while the adenosine triphosphate (ATP)/Pi ratio was decreased. The pH decreased from 7.02+/-0.03 to 6.93+/-0.04 during exercise and returned to control level (7.09+/-0.08) only during the second recovery period. These results suggest that the masseter muscle is characterized by high ATP turnover and, therefore, high oxidative phosphorylative activity in agreement with its constitution of predominantly fatigue resistant type I fibers.     

Metabolic response to halothane in piglets susceptible to malignant hyperthermia: an in vivo 31P-NMR study

Using in vivo 31P-nuclear magnetic resonance spectroscopy, we studied the skeletal muscle metabolism of 17 anesthetized malignant hyperthermia-susceptible piglets and 25 control piglets during and after a halothane stress test. At rest, the phosphocreatine- (PCr) to-ATP ratio was 12% higher in the anesthetized piglets than in the control piglets, which may reflect a higher proportion of fast glycolytic fibers in the former. About 15 min of halothane administration sufficed to provoke onset of a reaction, which was characterized by a reciprocal drop in PCr and an increase in Pi with commencing intracellular acidosis. Halothane was withdrawn after a 20% drop in PCr. Within the next few minutes, intracellular pH dropped sharply and phosphomonoesters (PME) accumulated excessively. ATP was observed to decrease in 8 of the 17 animals. Halothane inhalation provoked a switch of metabolism toward glycolysis. Accumulation of PME suggests a mismatch between glycogenolysis and glycolysis. Despite severe acidification, glycolysis was not completely halted. Recovery of PCr and Pi started approximately 5 min after halothane withdrawal, with a longer time constant for recovery of the former. PME and intracellular pH aberrations lingered and started to recover later. Lost ATP was never restored within the observed recovery period of approximately 20 min.     

Modification of ischemia and reperfusion damage of skeletal muscles with allopurinol: in vivo 31P MR spectroscopy of the posterior limb of the rat

The effect of Allopurinol on energy metabolism (re-utilisation of hypoxanthine) was studied in a in vivo skeletal muscle ischemia rat model by 31-P-MR spectroscopy. Allopurinol-treatment showed no benefit to the kinetics of PCr/(Pi + PCr) and ATP/(Pi + PCr). The role of re-utilisation of hypoxanthine has to be further investigated.     

The dynamic regulation of myocardial oxidative phosphorylation: analysis of the response time of oxygen consumption

Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O2 consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O2 consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O2 uptake during quick changes is measured with O2 electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O2 consumption in the mitochondria due to O2 diffusion and vascular transport. We correct for this transport delay by using the mass balance of O2, with all terms (e.g. O2 consumption and vascular O2 transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O2 and changes in cardiac O2 content. Experimental data on the response times of venous [O2] during step changes in arterial [O2] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O2 electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O2 consumption (tmito) to the step in heart rate is obtained. In isolated rabbit heart we found that tmito to heart rate steps is 4-12 s at 37 degrees C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (Pi) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and Pi do not change. Indeed, we found with 31P-NMR spectroscopy that phosphocreatine (PCr) and Pi change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant approximately 2.5 s) than mitochondrial O2 consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and Pi regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The tmito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O2 consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.     

The effects of extracellular pH and calcium manipulation on protein synthesis and response to anoxia/aglycemia in the rat hippocampal slice

Extracellular pH modulates the function of the N-methyl-D-aspartate (NMDA) receptor, which may influence pathophysiological responses to glutamate. While damage due to oxygen and glucose deprivation or glutamate exposure is attenuated by acidification of the incubating medium of cultured neurons, neuron damage is enhanced in vivo following ischemia in hyperglycemic animals. A persistent inhibition of protein synthesis (to less than 5% of normoxic levels) is a reliable index of damage to neurons both in vivo and in the rat hippocampal slice. We explored the influence of extracellular pH and calcium manipulation on protein synthesis inhibition and energy failure due to anoxia/aglycemia or exposure to N-methyl-D-aspartate in the rat hippocampal slice. Moderate acidification of the medium during anoxia/aglycemia did not reduce the damage to protein synthesis in hippocampal neurons (9% of normoxic levels) and did not alter basal ATP levels or the rate of ATP depletion during anoxia/aglycemia. However, when calcium levels were lowered during the acidification and following the anoxia/aglycemia, protein synthesis was almost completely protected (84% of normoxic levels). Calcium reduction itself also attenuated the protein synthesis inhibition due to anoxia/aglycemia (to 55.6% of normoxic controls), but the protection was not as complete. In contrast, moderate acidification of the medium significantly reduced the damage to protein synthesis due to a brief exposure to NMDA (37% of control with NMDA, 78.9% of control with acidification during NMDA), even in the presence of extracellular calcium. Alkalinization of the medium exacerbated the protein synthesis inhibition following anoxia/aglycemia, and significantly reduced basal ATP levels (to 52% of normoxic control levels). Thus, pHo changes influence neuronal metabolism and response to anoxia/aglycemia. In addition, while acidification can reduce the excitotoxic damage caused by direct exposure to NMDA, it cannot reduce damage due to anoxia/aglycemia unless calcium is lowered concomitantly. Thus, both NMDA receptor activation and calcium are involved in the damage due to oxygen and glucose deprivation in the slice.     

Screening a peptidyl database for potential ligands to proteins with side-chain flexibility

OBJECTIVE: Microdialysis and 31P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. METHODS: 31P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n = 9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n = 6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia-reperfusion (3 x 5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n = 8). RESULTS: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71 +/- 5 vs 43 +/- 8%, P < 0.05) and end-diastolic pressure (14 +/- 3 vs 29 +/- 4 mmHg, P < 0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (Pi), H+ or free Mg2+ during global ischemia, but improved recoveries of PCr, Pi, and delta GATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase, (5'-AMP), and in allosteric modulators of this enzyme (Mg2+, H+, Pi, ADP, ATP). CONCLUSIONS: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia-reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [Pi] and delta GATP in this model.     

Anoxia and ischemia tolerance in turtle hearts

It has been known since ancient times that turtle hearts exhibit extraordinary tolerance to anoxia or ischemia. The mechanisms by which they accomplish this remain obscure. The most important adaptation in anoxic turtles is a rapid and dramatic decrease in metabolic rate. Nuclear magnetic resonance measurements indicate that painted turtle (Chrysemys picta) hearts respond to anoxia with a rapid decrease in phosphocreatine (PCr; to 50% of control) after which PCr remains constant for at least 4 h. ATP is defended and does not decrease while intracellular pH (pHi) decreases by 0.2 pH units early in anoxia and is then maintained constant. Softshelled turtles (Trionyx spinifer) have been demonstrated to be far more sensitive than painted turtles to anoxia in vivo. However, isolated hearts from softshelled turtles appear to be as anoxia tolerant as those of Chrysemys. During ischemia there is also little difference in cardic performance, high energy phosphates, or pHi between these two species. A peculiar feature of turtle hearts is an extremely high concentration of phosphodiesters (PDE). The role of cytosolic PDEs remains controversial but they may function as lysophospholipase inhibitors and thereby limit phospholipid turnover (Burt CT and Ribolow H, Comparative Biochemistry and Physiology, 108B: 11-20, 1994). Whether PDEs promote anoxia/ischemia tolerance is unknown but these stresses can result in membrane lipid dysfunction in mammals. Metabolic control, acid-base, and phospholipid homeostasis all play a role in anoxia and ischemia tolerance in turtle hearts. These physiologic processes are interdependent, and how they interact in these animals is unknown, but they are experimentally accessible by modern analytical methods.     

Baseline consideration of liposomal contrast agent. CNS transport by macrophages in experimental allergic encephalomyelitis

The purpose of this study was to investigate a specialized liposomal contrast agent for magnetic resonance imaging (MRI), as part of a program to examine infiltrating immune cells in lesions of experimental allergic encephalomyelitis (EAE). A potent investigational liposomal contrast agent, phosphatidylethanolamine-DTPA-gadolinium, was chosen which has been shown to remain tightly liposomal-associated, with long persistence in vivo. Europium (Eu3+), a fluorescent paramagnetic metal, was also utilized in these experiments in place of gadolinium. This material is avidly taken up by monocytes in vivo. Thirty-four animals received some form of liposomal material either before or during the opening of the blood-brain barrier (BBB). Twenty-seven Hartley guinea pigs were inoculated for EAE with homogenized brain and Complete Freunds Adjuvant (CFA) and seven control animals received CFA alone. Eighty-two percent of the experimental animals exhibited degeneration of the BBB with inflammation and edema in the brain, while all control animals had normal brain scans. T1-weighted MRI, performed to detect the presence of liposomal contrast material in experimental animals, was not different from untreated animals. Fluorescent microscopy revealed no characteristic changes associated with Eu3+ presence in the brains of treated or control animals. Therefore, it would seem that insufficient material crosses the disrupted BBB, either in free form or subsequent to macrophage ingestion, to be detected by MRI or fluorescent microscopic examination.     

Requirement of glycolytic substrate for metabolic recovery during moderate low flow ischemia

Low flow ischemia with stable hemodynamic function can result in partial metabolic recovery characterized by an increase in phosphocreatine (PCr). Prior data suggest that glycolytic production of adenosine triphosphate (ATP) may be critical for this recovery and that the ATP produced by oxidative phosphorylation alone may be insufficient. This study tested the hypotheses that, during moderate low flow ischemia, (a) metabolic recovery is dependent on glycolytic production of ATP, and, therefore, (b) a mitochondrial substrate such as pyruvate alone is inadequate to allow metabolic recovery. High energy phosphates, pH, and lactate release were measured during 2 h of moderate low flow ischemia. Hearts were perfused with either a glycolytic plus mitochondrial substrate (glucose, insulin and pyruvate) or a mitochondrial substrate alone (pyruvate). Flow reductions required to reduce PCr by approximately 8% resulted in stable and equal reductions of rate-pressure product in each group. PCr recovered fully during the ischemic period in control hearts with glycolytic substrate, associated with preservation of normal end-diastolic pressure, and increased lactate release during the first hour of ischemia. Reperfusion of these hearts restored hemodynamic function and increased PCr above baseline values. In contrast, the use of pyruvate alone as a substrate resulted in a progressive fall of PCr during ischemia, increased end-diastolic pressure, and no significant increase in lactate release. Reperfusion in these hearts restored hemodynamic function, but did not result in normalization of PCr. Both groups had significant reductions in ATP during ischemia. Recovery of PCr during ongoing moderate low flow ischemia is observed in the presence of mixed glycolytic and mitochondrial substrates (glucose, insulin and pyruvate) but is not observed with pyruvate as a sole mitochondrial substrate. These data support a critical role for glycolytic flux under these conditions, suggesting that ATP generated solely by oxidative phosphorylation is not sufficient to promote metabolic recovery or maintain diastolic function during moderate low flow ischemia.     

Assessment of T2- and T1-weighted MRI brain lesion load in patients with subcortical vascular encephalopathy

Previous cross-sectional studies in patients with subcortical vascular encephalopathy (SVE) have shown little or no correlation between brain lesion load and clinical disability, which could be due to the low specificity of T2-weighted MRI. Recent studies have indicated that T1-weighted MRI may be more specific than T2-weighted MRI for severe tissue destruction. We studied 37 patients with a diagnosis of SVE and 11 normal controls with standardised T1- and T2-weighted MRI. All patients underwent detailed clinical assessment including a neuropsychological test battery and computerised gait analysis. Both the T2- and T1-weighted total MRI lesion loads different between patients and controls different, particularly T1. The ratio of T2-/T1-weighted lesion load was lower in controls than in patients. There was no overall correlation of T1- or T2-weighted lesion load with clinical disability, but group comparison of patients with severe and mild clinical deficits showed different lesion loads. We suggest that T1- and T2-weighted MRI lesion loads demonstrate relevant structural abnormality in patients with SVE.     

Indicators of oxidative stress in aging rat brain. The effect of nerve growth factor

INTRODUCTION AND OBJECTIVE: Increased oxidative stress during ageing and the neurodegenerative disorders associated with this has been described. The central nervous system is particularly vulnerable to oxidative damage because of its high energy requirements, high oxygen consumption, high tissue concentration of iron and relatively low levels of some antioxidant systems. Treatment with neurotrophic factors may reverse neurone deterioration and stimulate cholinergic activity in aged rats. It may have a similar neuroprotector effect against damage due to ischaemic reperfusion, hypoglycaemia, inflammation and other pathological conditions involving oxidative stress. In this study we determined some indicators of oxidative stress in rat brains during ageing and evaluated this in response to a plan of treatment with murine nerve growth factor (FCN) for 38 days. MATERIAL AND METHODS: Biochemical techniques were used for determination of oxidative stress indicators. RESULTS AND CONCLUSIONS: We found that with age there was a significant increase in phospholipase A2 and superoxide dysmutase activity and concentration of hipoperoxidases, whilst the concentration of reduced glutathion fell. Catalase activity increased in the hippocampal and striate regions and decreased in the cortex and septal area. There was less oxidative stress in rats treated with FCN. In view of our results, we conclude that the level of oxidative stress increases with ageing, with significant differences between areas of the brain. The region most vulnerable to damage from species reactive to oxygen was the hippocampus, and the protective effect of FCN may be related to potentiation of antioxidant defenses.     

Ecotoxicology and landscape planning

Relationships between pH and the concentrations of phosphocreatine (PCr), inorganic phosphate (Pi), and lactate during ischemic exercise depend on passive buffering, proton consumption as a consequence of net PCr breakdown, the control of glycogenolysis, (particularly in relation to the concentration of Pi, a substrate of glycogen phosphorylase that is produced by net PCr breakdown), and the creatine kinase equilibrium. The author analyzes the implications of these relationships for the interpretation of 31P-magnetic resonance spectroscopic data and for the control of glycogenolysis. For realistic adenosine diphosphate (ADP) concentrations, given the constraints of the creatine kinase equilibrium, the pH must be near-linear with lactate, with an apparent buffer capacity (i.e., the ratio of lactate accumulation to pH change) that is nearly twice the true buffer capacity (i.e., the ratio of net proton loading to pH change). The implications for glycogenolytic control depend on adenosine triphosphate (ATP) turnover, but an upper limit of activation of glycogen phosphorylase (i.e., the amount of the a form) that would permit no increase in ADP concentration can be calculated. Phosphorylase activation during ischemic exercise seems approximately proportional to the power output, consistent with calcium stimulation of phosphorylase b kinase. In simulations, ADP concentration is highly sensitive to this proportionality, as (unlike in purely oxidative exercise) ADP concentration is not known to participate in any closed feedback loops in ischemic exercise.     

Effect of DL-butylphthalide (NBP) on mouse brain energy metabolism in complete brain ischemia induced by decapitation

The effects of NBP on gasping and brain energy metabolism after complete brain ischemia in mice subjected to decapitation were investigated. The levels of ATP, phosphocreatine (PCr) and lactate were determined by the method of Lowry. The data indicated that NBP at 112.5 or 250 mg.kg-1 sc can significantly prolong the duration of gasping and at the dose of 150 or 200 mg.kg-1 sc reduce the level of lactate and increase the levels of ATP and PCr after complete brain ischemia. The results suggest that NBP may have brain protective action and improve ischemic brain energy metabolism.     

The in vivo relationship between blood flow, contractions, pH and metabolites in the rat uterus

Little is known about the relationship between smooth muscle contractile activity and its blood supply. We have therefore investigated this in the rat uterus, using laser-Doppler flow measurement and intra-uterine pressure recordings. We found an inverse linear relationship between flow and contractile activity. There was no evidence for a critical level of flow, above which function is maintained and below which it declines; even small reductions in blood flow decreased uterine force. Force was rapidly restored upon reperfusion. Reactive hyperaemia was absent from all but 6 of the 41 preparations studied. We used 31P nuclear magnetic resonance (NMR) spectroscopy to measure concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi) and intracellular pH (pHi) simultaneously with force and flow. Reductions in flow were associated with significant reductions in [ATP], [PCr] and pHi, and an increase in [Pi]. These changes were related to flow significantly and linearly and their effects on force may be additive. These data show that uterine smooth muscle is closely dependent upon its blood supply for maintaining both normal force production and metabolite levels. Consequently, even small decrements in flow may have deleterious functional effects.     

A study of the precision of confocal, ratiometric, Fura-2-based [Ca2+] measurements

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonia's effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chloride on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1-2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example, ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphological changes were observed 48 h following treatment in C6-glioma cells and at 9-10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.     

A comparison of AMP degradation in the perfused rat heart during 2-deoxy-D-glucose perfusion and anoxia. Part I: The release of adenosine and inosine

AMP degradation is studied in two models of the Langendorff-perfused rat heart which generate a large release of purines: the 2-deoxy-D-glucose (2DG)-perfused heart and the anoxic heart. In the 2DG model, mitochondrial energy generation is quasi-normal, despite a very low ATP concentration. Furthermore, inorganic phosphate (Pi) concentration is low, an important difference with anoxia where Pi is very high, up to 82 mM. Coronary release of purines is measured by high performance liquid chromatography, and myocardial metabolite content by 31P nuclear magnetic resonance spectroscopy. In the 2DG-perfused hearts with glucose or acetate, the purine release consists nearly exclusively of inosine [up to 130 nmol/(min x gww)] while adenosine is less than 1 nmol/(min x gww). A possible interpretation is that AMP degradation proceeds mainly through deamination to inosine monophosphate by AMP deaminase (the IMP pathway). In contrast, the purine release in anoxia (100% N2) contains comparable quantities of adenosine and inosine [respectively 30 and 20 nmol/(min x gww)], indicating that part of AMP is dephosphorylated directly to adenosine. Comparison with the 2DG model suggests that the release of adenosine in the anoxic heart is a result of inhibition of AMP deaminase by Pi.     

Role of oxygen in limiting respiration in the in situ myocardium

1H NMR has now detected the proximal histidyl N delta H myoglobin (Mb) signal from the myocardium in situ. Upon ligation of the left anterior descending coronary artery in the rat myocardium, the deoxy Mb signal appears at 78 ppm During dopamine infusion at up to 80 micrograms/kg/min, the heart rate pressure product (RPP) increases by a factor of 2, the phosphocreatine (PCr) decreases by 17%, and the ratio of the change in inorganic phosphate over PCr (delta Pi/PCr) increases by 0.2. However, no deoxy myoglobin signal is detected. Oxygen availability does not appear to limit oxygen consumption nor oxidative phosphorylation under dopamine enhanced work state in myocardium.     

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.