Rates Of Mitochondrial Oxidation Research Articles

Beta-hydroxybutyrate increases reactive oxygen species in late but not in early postimplantation embryonic cells in vitro.

Embryonic dysmorphogenesis has been blocked by antioxidant treatment in vivo and in vitro, suggesting that embryonic excess of reactive oxygen species (ROS) has a role in the teratogenic process of diabetic pregnancy. We report that the basal levels of ROS in dispersed rat embryonic cells in vitro, as determined by fluorescence of dichlorofluorescein (DCF), were not different in cells from control and diabetic pregnancy at day 10 or 12. Beta-hydroxybutyrate (beta-HB) and succinic acid monomethyl ester both augmented DCF fluorescence in cells from day 12 embryos of normal and diabetic rats but not from day 10 embryos. Cells of day 10 and day 12 embryos from normal and diabetic rats responded to increasing glucose concentrations with a dosage-dependent alleviation of DCF fluorescence. Day 10 embryonic cells exhibited high glucose utilization rates and high pentose phosphate shunt rates, but low mitochondrial oxidation rates. Moreover, in vitro culture of embryos between gestational days 9 and 10 in the presence of 20% oxygen induced an increased and glucose-sensitive oxidation of glucose compared with embryos not cultured in vitro. At gestation day 12, however, pentose phosphate shunt rates showed a decrease, whereas the mitochondrial beta-HB oxidation rates were increased compared with those at gestation day 10. This was paralleled by a lower expression of glucose 6-phosphate dehydrogenase- and phosphofructokinase-mRNA levels at day 12 than at day 10. On the other hand, H-ferritin mRNA expression at day 12 was high compared with day 10. None of the mRNA species investigated were affected by the diabetic state of the mother. It was concluded that beta-HB-induced stimulation of mitochondrial oxidative events may lead to the generation of ROS at gestational day 12, but probably not at day 10, when only a minute amount of mitochondrial activity occurs. Thus our results do not support the notion of diabetes-induced mitochondrial oxidative stress before the development of a placental supply of oxygen.

Alterations in carnitine–acylcarnitine translocase activity and in phospholipid composition in heart mitochondria from hypothyroid rats

Changes in mitochondrial fatty acid metabolism may underlie the decline in cardiac function in the hypothyroid animals. The effect of hypothyroidism on fatty acid oxidation, carnitine–acylcarnitine translocase activity and lipid composition in rat heart mitochondria has been examined. Rates of mitochondrial fatty acid oxidation as well as carnitine-carnitine and carnitine–palmitoylcarnitine exchange reactions were all depressed in heart mitochondria isolated from hypothyroid rats. Kinetic analysis of the carnitine–carnitine exchange reaction showed that the hypothyroid state affects the V max of this process, while having no effect on the K m value. Heart mitochondrial inner membrane lipid composition was significantly altered in hypothyroid rats. Cardiolipin, particularly, was found to decrease (by around 36%). Alterations in fatty acid pattern of mitochondrial inner membrane preparations from hypothyroid rats were also found. The effects of the hypothyroid state on fatty acids oxidation, carnitine translocase activity and phospholipid composition were completely reversed by following treatment of hypothyroid rats with thyroid hormone. A lower cardiolipin content in the mitochondrial inner membrane offers a plausible mechanism to explain the decline in the translocase activity in hypothyroidism.

Deficit of brain and skeletal muscle bioenergetics and low brain magnesium in juvenile migraine: an in vivo 31P magnetic resonance spectroscopy interictal study.

We used phosphorus magnetic resonance spectroscopy (31P MRS) to investigate in vivo the brain and skeletal muscle energy metabolism of 15 children with migraine with aura in interictal periods. Brain 31P MRS disclosed low phosphocreatine and high inorganic phosphate contents, and high intracellular pH in all patients. Calculated [ADP] and the relative rate of mitochondrial oxidation were higher in the brain of patients than in control subjects, whereas the phosphorylation potential was lower. Brain intracellular free Mg2+ concentration was reduced by 25% in patients. Abnormal skeletal muscle mitochondrial respiration was also disclosed in 7 of 15 patients as shown by the slow rate of phosphocreatine postexercise recovery. The multisystem bioenergetic failure found in patients with juvenile migraine is comparable to that found in adults with different types of migraine.

The Effects of the Ergosteroid 7-Oxo-dehydroepiandrosterone on Mitochondrial Membrane Potential: Possible Relationship to Thermogenesis

Administered 3β-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) is more effective than 3β-hydroxyandrost-5-en-7-one (DHEA) as an inducer of liver mitochondrialsn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in rats. Like DHEA, the 7-oxo metabolite enhances liver catalase, fatty acylCoA oxidase, cytosolicsn-glycerol-3-phosphate dehydrogenase, mitochondrial substrate oxidation rate, and the reconstructedsn-glycerol 3-phosphate shuttle. The mitochondrial adenine nucleotide carrier is diminished by thyroidectomy and is restored to normal activity by administering 7-oxo-DHEA. The relationship between respiratory rate and proton motive force across the mitochondrial membrane was measured in the nonphosphorylating state. When treated with increasing concentrations of respiratory inhibitors liver mitochondria from rats treated with 7-oxo-DHEA or thyroid hormones show a more rapid decline of membrane potential than do normal liver mitochondria. Thus 7-oxo-DHEA induces an increased proton leak or slip as has been reported for the thyroid hormone by M. D. Brand [(1990)Biochem. Biophys. Acta1018, 128–133]. This process may contribute to the enhanced thermogenesis caused by ergosteroids as well as by thyroid hormones.

L-Carnitine Stimulation of Mitochondrial Oxidative Phosphorylation Rate in Isolated Rat Skeletal Muscle Mitochondria

l-carnitine effects on mitochondrial adenosine triphosphate (ATP) production rate were investigated in mitochondria isolated from rat extensor digitorum longus (EDL) muscle. Kinetic parameters, apparent Michaelis constant for free adenosine diphosphate (ADP) (K m) and apparent maximal ATP production rate (V max), were determined in absence and presence of l-carnitine. Although the K m remained unchanged in response to l-carnitine addition when pyruvate + malate was oxidized, the affinity of isolated mitochondria towards ADP was decreased when pyruvate + palmitoylcarnitine + malate was used as substrate. As a result of l-carnitine addition, a slight non-significant ( P < 0.07) increase in V max was observed with pyruvate + malate while the increase reached the level of significance with pyruvate + palmitoylcarnitine + malate. Moreover, a positive correlation was obtained when mitochondrial ATP production rate was plotted against the difference between values measured with and without l-carnitine. Our data suggest that addition of l-carnitine to isolated rat skeletal muscle mitochondria can stimulate mitochondrial oxidative phosphorylation rate under conditions where the inhibitory effect of acetyl-CoA accumulation on pyruvate dehydrogenase complex activity is optimized. A change in the control of mitochondrial ATP flux by pyruvate dehydrogenase (PDH) complex might also occur with increasing mitochondrial ATP production rate, reinforcing the l-carnitine effects.

Activity of hepatic fatty acid oxidation enzymes in rats fed α-linolenic acid

The activity of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in α-linolenic acid ( α-18:3) was compared to that in rats fed safflower oil rich in linoleic acid (18:2) and a saturated fat (palm oil). Palm and safflower oils were essentially devoid of α-18:3. The palmitoyl-CoA oxidation rates both in mitochondrial and peroxisomal pathways in liver homogenates were significantly higher in rats fed linseed oil than in those fed palm and safflower oils. Among rats fed diets containing palm oil, safflower oil, fat mixtures composed of safflower and perilla oils (2:1, w/w and 1:2, w/w), and perilla oil, mitochondrial and peroxisomal fatty oxidation rates increased with increasing dietary levels of perilla oil. Compared to palm and safflower oils, dietary α-18:3 either in the form of linseed or perilla oils profoundly increased the activity of carnitine palmitoyltransferase, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase. Smaller but significant increases by dietary α-18:3 of the activity of acyl-CoA dehydrogenase, enoyl-CoA hydratase, and Δ 3, Δ 2-enoyl-CoA isomerase were also observed. Unexpectedly, dietary α-18:3 greatly reduced the activity of 3-hydroxyacyl-CoA dehydrogenase. Compared to palm oil, dietary polyunsaturated fats significantly reduced the activity of fatty acid synthetase and glucose-6-phosphate dehydrogenase to the same levels. The activity of pyruvate kinase was significantly higher in rats fed palm oil than in those fed polyunsaturated fats. The extent of reduction was more prominent with polyunsaturated fats containing α-18:3 than with safflower oil devoid of α-18:3. Thus, compared to linoleic acid and saturated fatty acids, dietary α-18:3 caused characteristic changes in the activity of hepatic enzymes in fatty acid and glucose metabolism in rats.

Glycerol is not an inhibitor of mitochondrial citrate oxidation by Aspergillus niger.

We have re-assessed the hypothesis that an accumulation of intracellular glycerol triggers the accumulation of citric acid by Aspergillus niger by inhibiting the activity of the mitochondrial NADP-dependent isocitrate dehydrogenase isoenzyme. To this end, we have incubated mycelia of A. niger with 0.5 M glycerol, which resulted in a maximal intracellular glycerol pool level of 0.92 M, comparable to that determined during the early phase of citrate accumulation. This addition affected neither the uptake of [1,5-14C] citrate from the medium nor the rate of the subsequent release of 14CO2 by the mycelia, indicating no effect on citrate oxidation. Mitochondria isolated from mycelia previously loaded with 0.5 M glycerol contained 8% of the total mycelial glycerol. They released 14CO2 from exogenously added [1,5-14C]citrate at the same rate as mitochondria isolated from mycelia not loaded with 0.5 M glycerol. The addition of glycerol had no effect on the activity of the purified NADP-specific isocitrate dehydrogenase, but appeared to inhibit the activity in crude cell-free extracts of A. niger. We conclude that the intracellular accumulation of glycerol does not affect the rate of mitochondrial citrate oxidation and is therefore, in contrast to previous claims, not a trigger of citrate accumulation.

Stimulation of the activities of hepatic fatty acid oxidation enzymes by dietary fat rich in alpha-linolenic acid in rats

The activities of hepatic fatty acid oxidation enzymes in rats fed perilla oil rich in alpha-linolenic acid (alpha-18:3) were compared with those fed saturated fats or safflower oil (the mixture of safflower oil and olive oil, 94:8, w/w) containing the same amount of polyunsaturated fatty acids with perilla oil exclusively as linoleic acid (18:2). When the rats were fed the diets containing 15% coconut, safflower, and perilla oils for 1 week, the rate of mitochondrial and peroxisomal oxidation of palmitoyl-CoA (16:0-CoA) in the liver homogenates was the highest in rats fed perilla oil. Among the rats fed the diets containing 15% palm, safflower, and perilla oils for 2 weeks, the rates of mitochondrial and peroxisomal oxidations of 16:0-, 18:2-, and alpha-18:3-CoAs were the highest in rats fed perilla oil, and the rate of oxidation of alpha-18:3-CoA by both pathways was higher than those of other acyl-CoAs in all groups. Dietary perilla oil relative to palm and safflower oils significantly increased the activities of carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, and 2,4-dienoyl-CoA reductase. The substrate specificity of carnitine palmitoyltransferase appeared to be responsible for differential rates of the mitochondrial oxidation of acyl-CoAs. The substrate specificity of acyl-CoA oxidase did not account for the preferential peroxisomal oxidation of alpha-18:3 relative to 18:2. The preferential mitochondrial and peroxisomal beta-oxidation of alpha-18:3-CoA relative to 16:0- and 18:2-CoAs was also confirmed in rats fed laboratory chow irrespective of the substrate/albumin ratios in the assay mixture. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed a diet rich in alpha-18:3.

2901 [ 11C]Acetylcarnitine as a probe for approaching the mechanisms of fatigue sensation

The reversibility of the carnitine-acetyl-CoA transferase and the carnitine-acetyl carnitine translocase system was studied in mitochondria isolated from rat heart muscle. Incubation of mitochondria with external acetyl-carnitine raised the levels of mitochondrial matrix acetyl-CoA and decreased free CoA. Total CoA and long chain acyl-CoA levels were not affected. A maximum increase in matrix acetyl-CoA was achieved with an acetyl-carnitine concentration of 2.5 mm. When free carnitine was added to mitochondria preloaded with acetyl-CoA, a rapid decrease in matrix acetyl-CoA and rise in free CoA occurred illustrating that matrix acetyl-CoA can transfer acetyl units to external carnitine. Incubation of mitochondria with both acetyl-carnitine and free CoA resulted in a large rise in external acetyl-CoA suggesting that the carnitine-acetyl-CoA transferase system is capable of producing acetyl-CoA in the cytosol. These data support the hypothesis that the carnitine-acetyl-CoA transferase and carnitine-acetyl carnitine translocase system provides the communication between the mitochondrial matrix and cytosolic spaces for coupling rates of cytosolic fatty acid activation with mitochondrial oxidative rates.

Testosterone stimulates the biosynthesis of m-aconitase and citrate oxidation in prostate epithelial cells

Mitochondria (m-)aconitase is a rate-limiting regulatory enzyme in prostate epithelial cells which minimizes citrate oxidation by these cells. This unique metabolic characteristic is responsible for the ability of the prostate to accumulate and secrete extraordinarily high levels of citrate. Testosterone is a major regulator of prostate growth and function, and stimulates citrate oxidation. Therefore, an important action of testosterone might be its stimulation of m-aconitase in prostate epithelial cells. Studies were conducted with rat ventral prostate (VP) epithelial cells to establish the effect of testosterone on the level of m-aconitase and corresponding citrate oxidation. Physiological concentrations (10 −7–10 −10 M) of testosterone in vitro markedly increased the level of m-aconitase in freshly prepared isolated prostate epithelial cells. This increase was apparent within 3 h of exposure to the hormone. The stimulatory effect of testosterone on m-aconitase was abolished by actinomycin D and by cycloheximide. Both the level of m-aconitase enzyme and the level of m-aconitase activity were similarly increased by testosterone treatment. Correspondingly, testosterone increased the rate of mitochondrial citrate oxidation while having no effect on the rate of isocitrate oxidation, thereby demonstrating that the action of testosterone is specifically targeted at the m-aconitase reaction. In vivo studies revealed that castration markedly decreased and testosterone administration increased the m-aconitase level of prostate epithelial cells. In contrast, neither liver nor kidney m-aconitase level was altered by castration. These studies demonstrate that testosterone regulates the biosynthesis of m-aconitase in prostate epithelial cells. It appears that this is a cell-specific effect since neither kidney nor liver m-aconitase was affected. The studies reveal that prostate epithelial cells contain a constitutive and an androgen-induced m-aconitase component; whereas, kidney and liver contain a constitutive but no androgen-induced m-aconitase. Unlike essentially all other cells, m-aconitase is a regulatory and regulated enzyme in prostate epithelial cells.

Time course of oxidative phosphorylation in liver mitochondria of chickens fed on high-protein diet.

1. Progressive alterations in oxidative phosphorylation of liver mitochondria were followed for 14 d in growing chickens fed on either semi-purified low (7%) or high (61%) protein-energy diet. Hepatic mitochondrial oxidative phosphorylation rates were assessed polarographically with pyruvate + malate as substrates. 2. The ADP:O values were reduced significantly 4 d after the feeding of a high-protein-energy diet, when compared with those in chickens fed on a low-protein-energy diet, whereas the state 3 oxidation rates in chickens fed on a high-protein-energy diet from day 6 to 14 were significantly lower than those in low-protein-fed chickens. 3. No changes in sensitivity of mitochondrial ATPase activity to oligomycin, expressed as % of total ATPase activity, were observed among chickens fed for 21 d on diets with various protein concentrations though the FoF1-ATPase activity, expressed per mg protein, tended to decrease in chickens fed on high-protein-energy diet. 4. These results suggest that the reduced ADP:O values for liver mitochondria in the high-protein-fed chickens may not be involved in the degrees of integrity of the FoF1-ATPase.

Ions, transport, and energetics in normal and diseased skeletal muscle

Magnetic resonance spectroscopy (MRS) has highlighted the relationship between intracellular ionic homeostasis and the control of muscle energetics. In skeletal muscle, the oxidative rate of ATP synthesis is largely controlled by ADP, the concentration of which is determined by the creatine kinase equilibrium that includes the concentration of H+. At the onset of aerobic dynamic exercise, ATP is maintained largely by glycolysis, producing lactic acid and PCr breakdown. Vasodilation follows and ATP synthesis becomes predominantly oxidative. During recovery, PCr resynthesis gives a measure of mitochondrial function, and pH recovery reflects the Na+:H+ antiport activity. Dynamic31P MRS measurements can be used to derive quantitative information about the processes (fluxes and concentrations) described above. In diseased muscle (e.g., mitochondrial myopathy, dystrophy, hypertension) specific changes are observed in some of the control functions, ionic fluxes, or mitochondrial oxidative rates.

Alteration of Pyruvate Metabolism in African Trypanosomes During Differentiation from Bloodstream into Insect Forms

In the presence of glucose and ample oxygen, insect form African trypanosomes release pyruvate more than 100-fold more slowly than do bloodstream forms. This rate decrease could not be accounted for simply by an increased mitochondrial pyruvate oxidation rate as inhibiting mitochondrial respiration increases pyruvate efflux to rates only 2-3% of that observed for bloodstream form trypanosomes. Alternatively, decreased pyruvate efflux from insect form trypanosomes could not be accounted for by decreased pyruvate transporter activity, which, surprisingly, was nearly as high in insect form trypanosomes as reported by us earlier for bloodstream forms (J. P. Barnard, B. Reynafarje, and P. L. Pedersen (1993) J. Biol. Chem. 268, 3654-3661). Rather, the low pyruvate efflux rate appears to be due primarily to reduced levels of the enzyme pyruvate kinase, which, in contrast to conclusions of an earlier study, is readily detected in insect form trypanosomes in the absence of added activators at an activity level about 4% of that found in bloodstream forms. Insect form pyruvate kinase seems to be located in the cytosol and exhibits kinetic profiles and constants nearly identical to those reported by us earlier for the bloodstream form enzyme (J. P. Barnard, and P. L. Pedersen (1988) Mol. Biochem. Parasitol. 31, 141-148). It is suggested that the reduced levels of pyruvate kinase, and hence the reduced pyruvate efflux rates, in insect form trypanosomes result from down regulation of the gene encoding the cytosolic enzyme.

Oxygen dependent sulfide detoxification in the lugwormArenicola marina

The lugwormArenicola marina L. oxidizes entering sulfide to thiosulfate. After 8 h of normoxic incubations with sulfide concentrations of 0.2 to 1.0 mmoll-1 thiosulfate in the coelomic fluid amounted up to about 4 mmoll-1 whereas sulfite concentrations were 100-fold lower and no accumulation of sulfate in the coelomic fluid was found. The sulfide oxidation was highly oxygen dependent. An increase of oxygen partial pressure (\(P_{O_2 }\)) in the medium was followed by enhanced thiosulfate production and by a decrease of sulfide concentration in the coelomic fluid. Under normoxia, the sulfide oxidation rate was sufficient to compensate the influx of sulfide into the coelomic fluid when the sulfide concentration in the medium was below 0.33 mmoll-1. When external sulfide was raised beyond this level, sulfide up to 5 μmoll-1 in the coelomic fluid appeared. Succinate in the body wall tissue was low as long as no sulfide appeared in the coelomic fluid, indicating the maintenance of an aerobic metabolism. The oxidation of sulfide to thiosulfate was localized in the mitochondria of the body wall tissue. The oxygen consumption of mitochondria was stimulated by the addition of sulfide. The mitochondrial sulfide oxidation rate depended on the amount of mitochondrial protein and followed a Michaelis-Menten kinetic. An apparentKm of 0.68±0.29 μmoll-1 and aVmax of 41.9±22.3 nmol min-1 mg-1 protein was calculated. Sulfide was stoichiometrically oxidized to thiosulfate with 1 mol sulfide consuming 1 mol oxygen. Sulfide oxidation was not inhibited by sulfide concentrations as high as 100 μmoll-1. At low concentrations of cyanide or azide, when respiration without sulfide was already inhibited, sulfide oxidation could still be stimulated, tentatively indicating the existence of an alternative terminal oxidase. Specimens examined in the present study were collected near St. Pol de Leon, France, from 1989 to 1992.

Effects of C-methylated carnitine analogs on rates of mitochondrial fatty acid oxidation

Carnitine analogs containing one and/or two methyl substituents on the α-, β-, or γ-carbon were evaluated in isolated rat liver mitochondria for their effects on fatty acid oxidation. Their abilities to either support, in the absence of carnitine, or inhibit, in the presence of carnitine, carnitine-dependent fatty acid oxidation were determined by the conversion of radiolabeled [1- 14C]palmitic acid to acid-soluble radiolabeled products. None of the methylcarnitine analogs were observed to be significant inhibitors of palmitate oxidation at concentrations (1.0 mM) up to ten times that for icarnitine. The two diastereomers of d,l-4-methylcarnitine, however, were able to support palmilate oxidation in the absence of carnitine, and rates were roughly 40% of that obtained with equimolar (0.1 mM) l-carnitine.

Mitochondrial pyruvate transport in working guinea-pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 μM-1.2 mM α-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, α-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO 2 production from [1- 14C]pyruvate or [U- 14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 μmol/min per g dry mass at S 0.5 ≈ 120 μ M , which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of ≈ 32 μ mol/min per g dry mass at 37° C . Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 μM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O 2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H + symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

Effects of pH and Temperature on the Kinetics of Pyruvate Oxidation by Muscle Mitochondria from Rainbow Trout (Oncorhynchus mykiss)

We measured the kinetics of pyruvate oxidation by mitochondria of rainbow trout (Oncorhynchus mykiss) red muscle and the kinetics of pyruvate decarboxylation by partially purified pyruvate dehydrogenase (PDH) from the same tissue. The measurements were done at three temperatures (8°, 15°, and 22°C) and at two pH conditions, a stable pH and an adjusted pH, that is, a pH that mimics the cellular pH adjustments with body temperature (ΔpH/ΔT= -0.03°C⁻¹). At constant pH, a change in temperature from 15° to 8°C decreased the apparent Km of mitochondria for pyruvate. However, when pH covaried with temperature, the apparent Km did not change. The Q10 for the Vmax of pyruvate oxidation was 2.5 between 8° and 15°C and 0.89 between 15° and 22°C (adjusted pH). Temperature significantly affected the pyruvate kinetics of PDH when pH was maintained constant (7.8) but not when pH covaried with temperature. Thus, pH regulation would minimize the functional impact of changes in temperature for both PDH and mitochondria. Comparison of the pyruvate kinetics suggests that PDH partially determines the mitochondrial affinity for pyruvate as well as its sensitivity to temperature and pH. Between 8° and 15°C, the Q10 of the Vmax of PDH was lower than that of intact mitochondria, suggesting that PDH did not limit the maximal rates of mitochondrial pyruvate oxidation. The thermal sensitivity of mitochondrialpyruvate oxidation was lower than that of the aerobic scope of the trout.

Control of phosphocreatine resynthesis during recovery from exercise in human skeletal muscle.

Information about the control of mitochondrial function in skeletal muscle in vivo can be obtained from the relationship between the rate of mitochondrial oxidation and the intracellular concentrations of phosphorus metabolites, although the analysis is complicated by the constraints imposed by the creatine kinase equilibrium. The rate of phosphocreatine (PCr) recovery after exercise measured by 31P MRS is an estimate of net oxidative ATP synthesis. Analysing such data from normal and abnormal human muscle, we show that the approximately exponential recovery kinetics of ADP and PCr imply that the rate of PCr resynthesis has a hyperbolic dependence on [ADP] but remains approximately linear with respect to the concentration of orthophosphate (Pi) and therefore also [PCr] and [creatine]. Both kinds of relationship are consistent with experimental data from exercising animal muscle and also with data from isolated mitochondria which suggest kinetic control of mitochondrial ATP synthesis of [ADP]. These relationships are altered in proven mitochondrial disease. This analysis offers a way to quantify mitochondrial function and its abnormalities in vivo.

Effect of Temperature and Coronary Flow on the Metabolic and Mechanical Function of the Isolated Rat Heart

A number of cardiac metabolic intermediates, namely, adenosine triphosphate (ATP), H+, phosphocreatine (PCr), inorganic phosphate (Pi), adenosine diphosphate (ADP), and related functions of these intermediates, Gibbs' free energy of ATP hydrolysis (delta G) and phosphorylation ratio [ATP/(ADP.Pi)], are thought to adjust mitochondrial oxidative phosphorylation rates to conform to mechanical demand. The effects of hypothermia and altered perfusion pressure on these parameters were evaluated in 12 hearts from Sprague-Dawley rats perfused in the Langendorff mode. 31P-nuclear magnetic resonance (NMR) spectra were obtained at cardiac temperatures between 20 and 37 degrees C, and coronary perfusion pressures between 20 and 145 cm H2O. Coronary flow varied between 0.5 and 15 ml/min throughout this range of intervention. Heart rate (HR), left ventricular systolic pressure (LVSP), and specific volumetric coronary flow (SCF) were determined for each temperature and perfusion pressure. The product HR x LVSP directly correlated with perfusion pressure at all temperatures. The temperature dependence could be represented by an overall activation energy of 72.7 kJ/M. In the constant temperature experiment, SCF and HR x LVSP fell linearly with decreasing perfusion pressure. Quantitative evaluation of the relationship between cardiac function and the metabolic intermediates described above defined these intermediates as nonregulatory with the possible exception of H+.

Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid.

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.