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Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved.
A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effect of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemia pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.
Transsarcolemmal movement of inorganic phosphate in glucose-perfused rat heart: a 31P nuclear magnetic resonance spectroscopic study.
Fluxes of orthophosphate (Pi) across the sarcolemma may be important in myocardial metabolism, yet little is known of these in the intact heart. We used 31P NMR spectroscopy to measure net Pi fluxes from changes in the concentrations of Pi, phosphocreatine (PCr), ATP and "total phosphate" ([TP] = [PCr] + 3[ATP] + [Pi]) in the isolated perfused rat heart in response to a change in extracellular [Pi] from 2 mM to 0 mM. [Pi] decreased to 62% of control with a half-time of 6 min, while [TP] decreased with initial rate 20 mM/h, a measure of net Pi efflux. As [PCr] decreased to 83% of control, phosphorylation potential remained constant. Contractile function was unaffected. Reperfusion with 2 mM Pi reversed all changes, causing net Pi influx at 26 mM/h. Analysed according to a model of net Pi flux, these imply a sarcolemmal permeability rate constant of 13 per h. Insulin in the 2 mM Pi perfusion buffer caused a transient decrease in intracellular [Pi] to 59% of control, while [TP] increased, giving a net Pi influx of 12 mM/h and a permeability constant of 12 per h. [PCr] increased by 28% over 34 min. Thus, insulin caused Pi influx by stimulating Pi incorporation into organic phosphates, transiently decreasing intracellular [Pi] and increasing the outside-to-inside [Pi] gradient. The response to 0 mM and 2 mM Pi perfusion was unaffected by insulin. We conclude that Pi fluxes across the sarcolemma are substantial and may have implications for Pi-free tissue perfusion and clinical hypophosphataemia.
Rural population mixing and childhood leukaemia: effects of the North Sea oil industry in Scotland, including the area near Dounreay nuclear site.
OBJECTIVE: To determine if any excess of childhood leukaemia and non-Hodgkin's lymphoma was associated with certain striking examples of population mixing in rural Scotland produced by the North Sea oil industry. DESIGN: Details were traced for over 30,000 workers involved in the construction of the large oil terminals in the Shetland and Orkney islands in northern Scotland or employed offshore. Home addresses of the 17,160 Scottish residents were postcoded, integrated with census data, and then classified as urban or rural. Rural postcode sectors, ranked by proportion of oil workers, were grouped into three categories with similar numbers of children but contrasting densities of oil workers. The incidence of leukaemia and non-Hodgkin's lymphoma was examined in these rural (and also in urban) categories in the periods 1974-8, 1979-83 and 1984-8. SETTING: Scotland. SUBJECTS: Young people below age 25. RESULTS: A significant excess of leukaemia and non-Hodgkin's lymphoma was found in 1979-83 in the group of rural home areas with the largest proportion of oil workers, following closely on large increases in the workforce. The area near the Dounreay nuclear installation, where an excess of leukaemia is already well known, was within the rural high oil category. CONCLUSION: The findings support the infection hypothesis that population mixing can increase the incidence of childhood leukaemia in rural areas. They also suggest that the recent excess in the Dounreay-Thurso area is due to population mixing linked to the oil industry, promoted by certain unusual local demographic factors.
The β/α peak height ratio of ATP. A measure of free [Mg2+] using 31P NMR
From 31P NMR measurements made in vitro at 38 °C, I = 0.25, pH 5.75- 8.5, and calculated free [Mg2+] from 0 to 5 mM, we show that, within the physiological range of cytosolic free [Mg2+] from 0.25 to 1.5 mM, the chemical shift difference between the α- and β-ATP resonances, δ(αβ), changes by only 0.6 ppm. Consequently, we developed new formalisms from known acid and Mg2+ dissociation constants by which the observed chemical shift of P(i), δ(P(i)), and the peak height ratio of the β- and α-ATP resonances, h(β/α), could be related to free [Mg2+] by simultaneous solution. We found that h(β/α) changed 2.5-fold as free [Mg2+] varied from 0.25 to 1.5 mM, providing a more sensitive and accurate measure of free cytosolic [Mg2+]. In working rat heart perfused with glucose, free [Mg2+] was 1.0 ± 0.1 from h(β/α) and 1.2 ± 0.03 from measured [citrate]/[isocitrate] but 0.51 ± 0.1 from δ(αβ). Addition of ketone bodies to the perfusate decreased free [Mg2+] estimated from h(β/α), to 0.61 ± 0.02 and 0.74 ± 0.11 by [citrate]/[isocitrate] but the estimate from δ(αβ) was unchanged at 0.46 ± 0.04 mM. Such differences in estimated free [Mg2+] alter the apparent K(eq) of the creatine kinase reaction and hence the estimated cytosolic free [ΣADP].
Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphonate markers.
To quantify metabolite and cation concentrations using NMR spectroscopy, the volumes of intracellular and extracellular spaces must be known. We describe a simple 31P NMR spectroscopic method that employs dimethyl methylphosphonate (DMMP) as a marker of total water space and phenylphosphonate (PPA) as a marker of extracellular space to determine intracellular and extracellular space volumes in the isolated, perfused rat heart. In vivo and in vitro radiolabel studies were used to verify this method. The difference between the total and extracellular water spaces, determined as milliliters/heart, gave the intracellular volume and allowed direct calculation of myocardial creatine phosphate, ATP, and inorganic phosphate concentrations, which were 13.4 mM, 10.1 mM, and 3.4 mM, respectively, for the glucose-perfused rat heart. The extracellular volume decreased by 84% in hearts subjected to 28 min total, global ischemia and increased by 15% during reperfusion. The method described allows the determination of intracellular energy metabolite concentrations in perfused rat heart directly from a single, fully relaxed 31P NMR spectrum.
Changes in probe sensitivity during NMR spectroscopic studies of the perfused rat heart: a warning.
During NMR experiments on isolated, perfused rat heart, we observed a 57% change in probe sensitivity when the perfusion buffer surrounding the heart was replaced by either a low-ionic-strength mannitol solution or air and the heart was made ischemic. Such changes in sensitivity, should they go unrecognized, would result in substantial quantification errors.
Evidence from population mixing in British New Towns 1946-85 of an infective basis for childhood leukaemia.
Mortality from leukaemia under age 25 was studied in British New Towns to test the hypothesis that leukaemia represents a rare response to a much commoner (but unrecognised) infection, the transmission of which is facilitated when large numbers of people come together. The density of children was higher in the rural, but lower in the overspill, New Towns than in the areas from which their incomers originated. Residents of the rural New Towns had greater diversity of origin than those of the overspill towns of London and Glasgow. These two factors would encourage a greater rise in the postulated underlying infection in the rural towns, and in these a significant excess of leukaemia at ages 0-4 was found in 1946-65. In both sets of towns there was a significant deficit in other age groups consistent with immunising effects of the relevant infection. There are parallels with feline leukaemia virus infection, in which contrasting leukaemogenic and immunising effects occur in different social settings owing mainly to differences in intensity of viral exposure.
A detailed 3D model of the rabbit right atrium including the sinoatrial node, atrioventricular node, surrounding blood vessels and valves
We used multiple techniques to generate a three-dimensional anatomical model of the rabbit right atrium with the sinoatrial node (SAN) and atrioventricular node (AVN). The model includes the right atrium, SAN, AVN, part of right ventricle, aorta with aortic valve, superior vena cava, inferior vena cava, coronary sinus, tricuspid valve, part of mitral valve, fossa ovalis and central fibrous body. The tendon of Todaro and right and left sinoatrial ring bundles were highlighted as landmarks. © 2005 IEEE.
Temporal relation between energy metabolism and myocardial function during ischemia and reperfusion.
The purpose of the present investigation was to study the relation between energy metabolism and contractile function in the isovolumic guinea pig heart. 31P nuclear magnetic resonance spectroscopy was used to measure changes in the intracellular levels of creatine phosphate, ATP, inorganic phosphate, and pH during 2.43 min total global ischemia and 2.43 min reperfusion, with a time resolution of 9.7 s. From these data, cytosolic changes in the phosphorylation potential, [ATP]-to-[ADP] ratio, free-energy change of ATP hydrolysis, and concentration of free ADP were estimated. The simultaneous monitoring of functional and biochemical parameters allowed them to be directly correlated with respect to time and with respect to each other. No significant changes in ATP were detected at any time, but changes in all other biochemical data were highly correlated with changes in contractile function. Kinetic analysis, using a nonlinear least-squares fit of the experimental points, revealed that the changes in most parameters fitted monoexponential functions. Each parameter was ranked according to its half time, which revealed that the phosphorylation potential was the only metabolic parameter to change at a rate faster than loss of contractile function during ischemia, and all metabolic changes, with the exception of pH, led the recovery of contractile function during reperfusion, the most rapid change occurring in the free ADP concentration. It is concluded that the cytosolic phosphorylation potential controls the contractile function of the heart and that cytosolic free ADP is important in the control of mitochondrial oxidative phosphorylation.
A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1).
In this study we show that mitochondrial uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and thymus mitochondria can be ubiquitinylated and degraded by the cytosolic proteasome. Using a ubiquitin conjugating system, we show that UCP1 can be ubiquitinylated in vitro. We demonstrate that UCP1 is ubiquitinylated in vivo using isolated mitochondria from brown adipose tissue, thymus and whole brown adipocytes. Using an in vitro ubiquitin conjugating-proteasome degradation system, we show that the cytosolic proteasome can degrade UCP1 at a rate commensurate with the half-life of UCP1 (i.e. 30-72h in brown adipocytes and ~3h, in thymocytes). In addition, we demonstrate that the cytoplasmic proteasome is required for UCP1 degradation from mitochondria that the process is inhibited by the proteasome inhibitor MG132 and that dissipation of the mitochondrial membrane potential inhibits degradation of UCP1. There also appears to be a greater amount of ubiquitinylated UCP1 associated with BAT mitochondria from cold-acclimated animals. We have also identified (using immunoprecipitation coupled with mass spectrometry) ubiquitinylated proteins with molecular masses greater than 32kDa, as being UCP1. We conclude that there is a role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial UCP1. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Control of choline oxidation in rat kidney mitochondria.
Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173+/-64 microM and a Vmax of 0.4+/-0.1 nmol/min/mg mitochondrial protein (at 10 degrees C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 degrees C (125+/-6 pmol O2/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96+/-4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.
The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue.
We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.
Detection of UCP1 protein and measurements of dependent GDP-sensitive proton leak in non-phosphorylating thymus mitochondria.
Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.
Uncoupling protein 1 dependent reactive oxygen species production by thymus mitochondria.
We have previously shown that uncoupling protein 1 is present in thymus and has a role in T-cell development. As reactive oxygen species have been implicated in T-cell development, we set out to determine whether uncoupling protein 1 had the potential to regulate reactive oxygen species production in mitochondria isolated from thymus. This was achieved by inhibiting proton leak through uncoupling protein 1 using the purine nucleotide GDP and through ablation of uncoupling protein 1, measuring the amplex red sensitive reactive oxygen species production by mitochondria. In this work we demonstrate, for the first time, that uncoupling protein 1 has the potential to regulate reactive oxygen species production in thymus mitochondria. We also show that reverse electron transport is possible in thymus mitochondria respiring on succinate and glycerol-3-phosphate. The implications of this regulatory role for uncoupling protein 1 are discussed in the context of thymus function. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
Insulin, ketone bodies, and mitochondrial energy transduction.
Addition of insulin or a physiological ratio of ketone bodies to buffer with 10 mM glucose increased efficiency (hydraulic work/energy from O2 consumed) of working rat heart by 25%, and the two in combination increased efficiency by 36%. These additions increased the content of acetyl CoA by 9- to 18-fold, increased the contents of metabolites of the first third of the tricarboxylic acid (TCA) cycle 2- to 5-fold, and decreased succinate, oxaloacetate, and aspartate 2- to 3-fold. Succinyl CoA, fumarate, and malate were essentially unchanged. The changes in content of TCA metabolites resulted from a reduction of the free mitochondrial NAD couple by 2- to 10-fold and oxidation of the mitochondrial coenzyme Q couple by 2- to 4-fold. Cytosolic pH, measured using 31P-NMR spectra, was invariant at about 7.0. The total intracellular bicarbonate indicated an increase in mitochondrial pH from 7.1 with glucose to 7.2, 7.5 and 7.4 with insulin, ketones, and the combination, respectively. The decrease in Eh7 of the mitochondrial NAD couple, Eh7NAD+/NADH, from -280 to -300 mV and the increase in Eh7 of the coenzyme Q couple, Eh7Q/QH2, from -4 to +12 mV was equivalent to an increase from -53 kJ to -60 kJ/2 mol e in the reaction catalyzed by the mitochondrial NADH dehydrogenase multienzyme complex (EC 1.6.5.3). The increase in the redox energy of the mitochondrial cofactor couples paralleled the increase in the free energy of cytosolic ATP hydrolysis, delta GATP. The potential of the mitochondrial relative to the cytosolic phases, Emito/cyto, calculated from delta GATP and delta pH on the assumption of a 4 H+ transfer for each ATP synthesized, was -143 mV during perfusion with glucose or glucose plus insulin, and decreased to -120 mV on addition of ketones. Viewed in this light, the moderate ketosis characteristic of prolonged fasting or type II diabetes appears to be an elegant compensation for the defects in mitochondrial energy transduction associated with acute insulin deficiency or mitochondrial senescence.