Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic,ionic, contractile and electrophysiological studies

Summary The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37°C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22°C and 27°C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37°C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24±2.49 IU·g dry wt^-1, and the total amount of protein lost was 70.37±0.73 mg·g dry wt^–1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37°C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.     

Various divalent cations protect the isolated perfused pigeon heart against a calcium paradox

The protective effects of various divalent cations against the irreversible damage of myocardium, a phenomenon termed the Ca²⁺-paradox, were examined in the isolated perfused pigeon heart. All cations examined were added at a concentration of 200 mol l^–1 in the calcium-free medium. In hearts perfused with low calcium, upon normal calcium repletion, the maximal recovery of the contractile tension (in the 2nd minute) was approximately 115% and the recovery obtained at the end of reperfusion was 81.5% (compared to the equilibration period value). From the other divalent cations examined, the presence of cobalt, nickel, manganese or barium during calcium depletion powerfully protected the pigeon heart. Upon calcium repletion, the maximal recovery of contractile tension was approximately 60%, 76.5%, 100% and 85%, the recovery estimated at the end of reperfusion was 40%, 12%, 70% and 53%, and the resting tension estimated at the end of reperfusion was 2.69±0.18 g, 6.40±0.50 g, 1.20±0.10 g and 1.90±0.10 g for cobalt, nickel, manganese and barium, respectively. On the contrary, strontium exerted no protective effects. The protective effects were also indicated by reduced total protein and lactate dehydrogenase activity release into the effluent perfusate and maintenance of electrical activity. The effectiveness of the added divalent cations (with the exception of strontium) showed a strong dependence upon their ionic radius. The most potent inhibitors of this phenomenon in the pigeon heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanisms involved in the protective effects of these cations.Communicated by: G. Heldmaier     

D-3-Hydroxybutyrate metabolism in the perfused rat heart

Summary The utilization of D-3-HB and the production of acetoacetate by the perfused rat heart were investigated over a wide range of DL-3-HB concentrations. The rate of D-3-HB utilization is concentration dependent, and shows saturation kinetics. The oxidized amount of D-3-HB when D-3-HB as a sole substrate, accounts at a maximum for 50% of the total oxygen consumption, which suggest the contribution of the endogenous substrate as fuel source along with D-3-HB. The proportion of the D-3-HB consumed that is oxidized rather than released as acetoacetate increases from 70% to 93% as the concentration of D-3-HB falls from 6.99 mM to 0.30 mM.     

Effect of calcium depletion and subsequent repletion on parathyroids,parafollicular (C) cells and bone in the growing pig

Summary The ultrastructure of the chief cells of the parathyroid gland and thyroid parafollicular (C) cells and the morphology of bone in calcium depletion and subsequent repletion were examined in young growing pigs. A low calcium diet resulted in osteopenia, increased removal of the cartilaginous core, osteoclasia and osteocytic osteolysis. Subsequent repletion quickly returned bone to normal. In pigs fed the low calcium diet, there was a marked depletion of secretory granules but a striking increase in the number of microtubules in chief cells. Increasing the calcium content of the diet to normal quickly returned the ultrastructural appearance of chief cells to apparent normal. In the initial response to calcium repletion, chief cells exhibited large number of lysosomes and occasionally prominent paracrystalloid bodies. Electron microscopic examination of parafollicular (C) cells of the thyroid gland failed to reveal differences in ultrastructure between test and control pigs. These findings support the view that bone resorption following calcium deficiency may be the result of a secondary hyperparathyroidism rather than of calcium deficiency per se.Supported by U.S.P.H.S. Grant A.M. 12957 from the Division of Arthritis and Metabolic Diseases     

Effect of ischemia,calcium depletion and repletion,acidosis and hypoxia on cellular taurine content

Summary.  Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na⁺]_i. The data suggest that taurine efflux is regulated by [Na⁺]_i and cellular osmolality, but not by cellular pH. Received November 15, 2001 Accepted January 15, 2002 Published online October 3, 2002 Acknowledgements This study was supported with a grant from the Taisho Pharmaceutical Company. Authors' address: Dr. Stephen W. Schaffer, Department of Pharmacology, University of South Alabama, School of Medicine, Mobile, Alabama, U.S.A., E-mail: sschaffe@jaguarl.usouthal.edu     

Release of fatty acid-binding protein and long chain fatty acids from isolated rat heart after ischemia and subsequent calcium paradox

To obtain insight into the relation between the release of heart-type fatty acid-binding protein (H-FABP_c) and of long-chain fatty acids (FA) from injured cardiac tissue, rat hearts were Langendorff perfused according to the following scheme: 30 min normoxia, 60 min ischemia, 30 min reperfusion, 10 min Ca²⁺ free perfusion and finally 10 min Ca²⁺ repletion. During this protocol right ventricular (Q _rv ) and interstitial effluent samples (Q _i ) were collected at regular intervals. During reperfusion a total of 0.8±0.1 nmol H-FABP_c but no FA were detected in the effluents. However, during Ca²⁺ readmission, 45±4 nmol H-FABP_c (80–90% of total tissue content) was released with an initial (first 3 min) simultaneous release of FA (FA/H-FABP_c ratio 0.90±0.07 mol/mol). Thereafter, FA release continued at 10–15 nmol per min mainly inQ _rv while the rate of H-FABP_c release decreased. During Ca²⁺ repletion, tissue FA content raised rapidly from 168±20 to 1918±107 nmol/g dry weight. These findings suggest that after severe cardiac damage initially FA is released bound to H-FABP_c, whereas further FA release occurs in a non-protein bound manner.     

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C₃ compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C₃ compounds formed than on the actual rate of C₄ compound disposition.     

Alterations of the aryl hydrocarbon hydroxylase system during riboflavin depletion and repletion

The alterations of the microsomal aryl hydrocarbon hydroxylase system in mice during riboflavin depletion and repletion have been examined. During the development of riboflavin deficiency, there was a decrease in the activity of the flavoprotein NADPH-cytochrome c reductase accompanied by an increase in cytochrome P-450 concentration. The aryl hydroxylase activities of the deficient animals were only slightly lower than the controls when isolated microsomes were used for the assay and the extent of decrease was more pronounced when liver homogenates were used for the assay. Upon repletion of flavin to the deficient mice, there were sharp rises in both the NADPH-cytochrome c reductase and aryl hydroxylase activities and a moderate decrease in cytochrome P-450 concentration in the first 2 days. The aryl hydroxylase activity of the microsomes of deficient mice can be elevated by preincubating with FAD or FMN, suggesting that the flavin coenzyme and hence the holo-reductase is rate limiting for the overall hydroxylation. During the recovery from riboflavin deficiency, the aryl hydroxylase can be induced by 3-methylcholanthrene to a greater extent than with the controls. The implications of these observations are discussed.     

Measurements of fatty acid and carbohydrate metabolism in te isolated working rat heart

The isolated working rat heart is a useful experimental model which allows contractile function to be measured in hearts perfused at physiologically relevant workloads. To maintain these high workloads the heart is required to generate a tremendous amount of energy. In vivo this energy is derived primarily from the oxidation of fatty acids. In many experimental situations it is desirable to perfuse the isolated working heart in the presence of physiologically relevant concentrations of fatty acids. This is particularly important when studying energy metabolism in the heart, or in determining how fatty acids alter the outcome of myocardial ischemic injury [1, 2]. The other major source of energy for the heart is derived from the oxidation of carbohydrates (glucose and lactate), with a smaller amount of ATP also being derived from glycolysis. Two byproducts of both fatty acid and carbohydrate metabolism are H2O and CO2. By labeling the glucose, lactate, or fatty acids in the perfusate with 3H or 14C the experimenter can quantitatively collect either 3H2O or 14CO2 produced by the heart. By using radioisotopes that are labeled at specific hydrogen or carbon molecules on the various energy substrates, and by knowing the specific activity of the radiolabeled substrate used, it is possible to determine the actual rate of flux through these individual pathways. This paper will describe the experimental protocols for directly measuring fatty acid and carbohydrate metabolism in isolated working rat hearts.     

Lipid-mediated impairment of normal energy metabolism in the isolated perfused diabetic rat heart studied by phosphorus-31 NMR and chemical extraction

The relationship between extracellular palmitate and the accumulation of long-chain fatty-acyl coenzyme A with that of high-energy phosphate metabolism was investigated in the isolated perfused diabetic rat heart. Hearts were perfused with a glucose/albumin buffer supplemented with 0, 0.5, 1.2 or 2.0 mM palmitate. ³¹P-NMR was used to analyze phosphocreatine and ATP metabolism during 1 h of constant-flow recirculation perfusion. At the end of perfusion, frozen samples were taken for chemical analysis of high-energy phosphates and the free and acylated fractions of coenzyme A and carnitine. Perfusion of diabetic hearts with palmitate, unlike control hearts, caused a time-dependent and concentration-dependent reduction in ATP, despite normal and constant phosphocreatine. Concentrations of acid-soluble coenzyme A, long-chain-acyl coenzyme A and total tissue coenzyme A were elevated in palmitate-perfused diabetic hearts, while the total tissue carnitine pool was decreased. Increases in long-chain-acyl coenzyme A correlated with the reduction in myocardial ATP. This reduction in ATP could not be adequately explained by alterations in heart rate, perfusion pressure or vascular resistance.     

The effect of fasting on D-3-hydroxybutyrate metabolism in the perfused rat heart

Summary The effects of fasting for 24 h and 48 h on D-3-hydroxybutyrate utilization and acetoacetate, L-lactate and pyruvate production by the isolated non-working perfused rat heart were investigated over a wide range of DL-3-HB concentrations. D-3-HB utilization is concentration dependent and shows saturation kinetics, D-3-HB oxidation is correlated with D-3-HB concentration. Acetoacetate production is proportional to DL-3-HB concentration. L-lactate production is proportional to DL-3-HB concentration up to 5 mM following a 24h fast and up to 10 mM after a 48h fast, but further increase in DL-3-HB concentration decreases the rate of lactate production. Fasting enhances D-3-HB utilization at 16 mM DL-3-HB by 16% and 25% in 24 h and 48 h fast respectively, but has no significant effect at lower concentration. Fasting has no effect on acetoacetate production. Fasting for 48 h doubled the half-saturation concentration (Ku) without significant change in the maximum rate of utilization (Vu) of D-3-HB.     

Effect of experimentally induced thyrotoxicosis on oxidative energy metabolism in rat heart mitochondria

Treatment of rats with T₃ resulted in a significant decrease in body weight, while the heart weight increased. T₄ treatment had less marked effect on body weights but resulted in decreased heart weights. Serum T₄ levels decreased significantly with simultaneous increase of T₃ level following T₃ treatment, whereas with T₄ treatment, levels of both T₄ and T₃ increased in the serum. Low doses of T₃ (0.5 μg ) caused decrease in mitochondrial protein content while high dose of T₄ (1 μg), caused significant increase in mitochondrial mass. The state 3 respiration rates were significantly depressed following T₃ and T₄ treatments, in a substrate specific manner with the effects being more pronounced with T₃; these responses with T₄ were dose-dependent for succinate and ascorbate + N,N,N′,N′-tetramethyl-p-phenylenedíamme. State 4 respiration rates also exhibited similar corresponding changes. ADP/O ratios were not changed but ADP-phosphorylation rates were decreased significantly particularly so with the T₃-treated animals. Treatment with T₃ also resulted in lowering of intramitochondrial cytochrome contents. Similar effects were seen also with higher doses of T₄. The results thus indicate that T_3- and T_4- thyrotoxicosis results in impaired energy metabolism in heart mitochondria.     

Proteome alterations of cortex and hippocampus tissues in mice subjected to vitamin A depletion

Vitamin A regulates the development and maintenance of the central nervous system. Studies of vitamin A depletion (VAD) and mutations of retinoid receptors in rodents have revealed a dysfunction of motor and cognitive abilities. However, the molecular mechanisms underlying these behavioral changes are not well understood. In this study, VAD mice were examined and abnormal motor behavior related to psychosis symptoms was found. With the use of two-dimensional gel electrophoresis (2-DE), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometric (MS) technologies, 44 and 23 altered protein spots were identified in the cortex and hippocampus, respectively, in VAD mice. By Western blot, the up-regulation of mitogen-activated protein kinase 1 (MAPK1) and proteasome subunit beta type 2 (PSMB2) in the cortex and that of dihydropyrimidinase-related protein 2 (DPYSL2) and PSMB2 in the hippocampus were observed in VAD mice. Bioinformatic analysis using DAVID revealed that altered proteins induced by VAD showed significant enrichment of (i) glycolysis, cytoskeleton, mitochondrion and glutamate metabolism in the cortex; and (ii) actin binding, dopamine receptor signaling and transmission of nerve impulse in the hippocampus. The up-regulations of DPYSL2, MAPK1 and PSMB2 may indicate the activated neuronal defensive mechanism in VAD brain regions, which may underlie the VAD-related psychosis behavior.     

Creatine kinase,energy-rich phosphates and energy metabolism in heart muscle of different vertebrates

Maximal activities of creatine kinase, pyruvate kinase and cytochrome oxidase and total concentrations of creatine and phosphorylated adenylates were measured in cardiac muscle of hagfish, eight teleost species, frog, turtle, pigeon and rat. The ratio of creatine kinase to cytochrome oxidase with cytochrome oxidase as a rough estimate of aerobic capacity and cellular “energy turnover”, was increased in myocardia of hagfish, turtle and crucian carp. These myocardia are likely to be frequently exposed to oxygen deficiency. In agreement with this, they possess a high relative glycolytic capacity as indicated by a high pyruvate kinase/cytochrome oxidase ratio. The creatine kinase/cytochrome oxidase ratio for the other myocardia varied within a factor of 2, except the value for cod myocardium which was below the others. Total creatine varied among species and was high in active species such as herring, pigeon and rat but also high in crucian carp. The variation in total concentration of phosphorylated adenylates was considerably less than the variation in total creatine. The high creatine kinase/ cytochrome oxidase ratio in myocardia likely to be challenged by hypoxia may represent an enhanced efficiency for both “spatial” and “temporal” buffering of phosphorylated adenylates to attenuate the impact of a depressed energy liberation. As to the differences in total creatine, this factor influences not only the cellular energy distribution but possibly also contractility via an effect on the free phosphate level.     

冷驯化条件下大绒鼠的产热和能量代谢特征

本文主要研究了冷驯化(5℃±1℃)条件下,大绒鼠(Eothenomys miletus)的能量收支、基础代谢率(BMR)、非颤抖性产热(NST)和肝脏线粒体呼吸.结果表明:随着冷驯化的进行,大绒鼠的体重、体温降低;摄入能、消化能、可代谢能增加;BMR和NST增加;肝脏线粒体呼吸状态Ⅲ呼吸先增加,28天后趋于平稳;线粒体状态Ⅳ呼吸先增加,28天后下降.说明在冷驯化条件下,大绒鼠采取适当降低体重和体温、增加能量摄入、增加BMR和NST产热的对策来维持能量平衡     

Comparative properties of bovine heart and liver rhodaneses and the regulatory role of the rhodaneses in energy metabolism

In previous studies on the rhodanese activity of bovine liver mitochondria, we have shown that in addition to activity observed in the soluble protein fraction, there is rhodanese activity that is bound to the mitochondrial membrane. The latter activity accounts for as much as 40% of the total and, in situ, is associated in a multiprotein complex that forms iron-sulfur centers. In the present studies, we have investigated the rhodanese activity of bovine heart muscle. We have found that the major part of this enzyme activity is localized in the mitochondria and, further, that at least 25% of the total rhodanese activity of heart mitochondria is membrane-bound. As in liver tissue, the heart activity at least in part is associated in a multiprotein complex that forms iron-sulfur centers. Upon purification of the heart rhodanese in the soluble protein fraction, there is a 10- to 30-fold decrease inK _m values for the standard assay substrates thiosulfate and cyanide ions. These observations are consistent with the interpretation that there are activated and deactivated (low activity) forms of the heart enzyme in crude extracts, but only the activated form survives purification. The present results, together with our recent finding that liver mitochondrial rhodanese is subject to phosphorylation, lend support to our proposal that the rhodaneses serve as converter enzymes which regulate the rate of electron transport through sulfuration of respiratory chain components. The rhodaneses, in turn, are controlled by protein kinases and the local ATP concentration.     

Alterations in lipid and calcium metabolism associated with seizure activity in the postischemic brain

Transient ischemia is known to lead to a long-lasting depression of cerebral metabolic rate and blood flow and to an attenuated metabolic and circulatory response to physiological stimuli. However, the corresponding responses to induced seizures are retained, demonstrating preserved metabolic and circulatory capacity. The objective of the present study was to explore how a preceding period of ischemia (15 min) alters the release of free fatty acids (FFAs) and diacylglycerides (DAGs), the formation of cyclic nucleotides, and the influx/efflux of Ca(2+), following intense neuronal stimulation. For that purpose, seizure activity was induced with bicuculline for 30 s or 5 min at 6 h after the ischemia. Extracellular Ca(2+) concentration (Ca(2+)(e)) was recorded, and the tissue was frozen in situ for measurements of levels of FFAs, DAGs, and cyclic nucleotides. Six hours after ischemia, the FFA concentrations were normalized, but there was a lowering of the content of 20:4 in the DAG fraction. Cyclic AMP levels returned to normal values, but cyclic GMP content was reduced. Seizures induced in postischemic animals showed similar changes in Ca(2+)(e), as well as in levels of FFAs, DAGs, and cyclic nucleotides, as did seizures induced in nonischemic control animals, with the exception of an attenuated rise in 20:4 content in the DAG fraction. We conclude that, at least in the neocortex, seizure-induced phospholipid hydrolysis and cyclic cAMP/cyclic GMP formation are not altered by a preceding period of ischemia, nor is there a change in the influx/efflux of Ca(2+) during seizure discharge or in associated spreading depression.     

中缅树鼩能量代谢的季节变化

小型哺乳动物的体重和产热特征的季节调节对其生存至关重要。为探讨中缅树鼩的能量代谢适应特征随季节的变化,采用耗氧量测定、食物平衡法、形态测量等方法,分别对其冬季和夏季的基础代谢率(BMR)、非颤抖性产热(NST)、体温、体重、蒸发失水、能量收支和消化道的长度和重量进行了测定。中缅树鼩冬季体温、体重、基础代谢率、NST、蒸发失水散热分别为37. 9℃ ± 0.14℃ ,126.1 ± 2.1 g,42. 94 ± 2.65 J/g· h,54. 97 ±2.14 J/ g·h,5. 69 ±0.33 J/ g·h;夏季体温、体重、基础代谢率、NST、蒸发失水散热分别为38.5℃ ± 0. 27℃ ,106.9 ±5.1 g,28. 69 ±3.06 J/ g·h,47.43 ± 2.45 J / g·h,7.12 ±0. 57 J/ g·h;中缅树鼩的每日摄入能、消化能、可代谢能冬季均比夏季显著增加,消化道特征冬季和夏季存在变化,随着温度降低、食物质量下降,小肠长度和重量增加。这些结果表明:中缅树鼩在冬季,通过增加体重、基础代谢率和NST、能量摄入、消化能和可代谢能,降低蒸发失水等方式应对季节性环境变化。代谢产热和消化生理调节在季节性适应过程中具有重要地位。     

Regulation of energy metabolism in liver

Energy metabolism in liver has to cope with the special tasks of this organ in intermediary metabolism. Main ATP-generating processes in the liver cell are the respiratory chain and glycolysis, whereas main ATP-consuming processes are gluconeogenesis, urea synthesis, protein synthesis, ATPases and mitochondrial proton leak. Mitochondrial respiratory chain in the intact liver cell is subject to control mainly by substrate (hydrogen donors, ADP, oxygen) transport and supply and proton leak/slip. Whereas hormonal control is mainly on substrate supply to mitochondria, proton leak/slip is supposed to play an important role in the modulation of the efficiency of oxidative phosphorylation.     

Osteoclast and its roles in calcium metabolism and bone development and remodeling

Osteoclasts are multinucleated cells responsible for bone resorption and play important roles in normal skeletal development, in the maintenance of its integrity throughout life, and in calcium metabolism. During bone resorption, the cytoskeleton of osteoclasts undergoes extensive reorganization, with polarization and formation of ruffled borders to secrete acid and formation of sealing zone to prevent leakage. The differentiation and function of osteoclasts are in turn regulated by osteoblasts, stromal cells, and bone. They are also subjected to negative feedback regulation by extracellular and intracellular calcium concentrations.