Significance of extracellular concentration of sodium ions in the protective effect during the "calcium paradox"]

Increasing of extracellular sodium concentration up to 200 mM diminishes heart damage under "calcium paradox". Phosphocreatine (10(-4) M) potentiates the effect of high sodium perfusion media; in this case myoglobin release from the myocardium is minimal (5-9% of control). An the same time, ATP and phosphocreatine concentrations and oxidation to phosphorylation coupling in mitochondria remain at a sufficiently high level. Elevation of osmotic pressure by the effect of 120 mM sucrose enhances heart damage under "calcium paradox" both in the presence and absence of phosphocreatine. The protective effects of superhigh (200 mM) sodium concentrations and phosphocreatine are completely reversed by strophanthin or decreasing K+ concentration down to 0.5 mM.     

Prevention of myocardial reperfusion injury by increasing artificial trans-membrane sodium gradient in "calcium paradox" and post-ischemic reperfusion

An effect of the high sodium gradient during "calcium paradox" and postischemic reperfusion has been studied. A decrease of Na/Ca exchange by high sodium gradient (200 mM NaCl in the perfusion solution) resulted in the reduction of myoglobin release from the heart during "calcium paradox". High sodium concentration solution (200 mM) increased protective effect of ATP during "calcium paradox". Exogenous phosphocreatine (100 mumol/mol) increased myoglobin release from the heart. During perfusion of the heart by high sodium concentration, phosphocreatine efficiently decreased myoglobin release from the heart during "calcium paradox". Exogenous ATP (as Na-pump activator) and high Na+ concentration solution (180 mM) prevented the LDH release from the myocardium, decreased ATP hydrolysis, inhibited Ca influx, maintained total adenine nucleotides, phosphate potential, energy charge of the cardiomyocytes.     

Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types

Total creatine (Cr(total) = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add Cr(total) to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [Cr(total)] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05-1 mM [(14)C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 +/- 8.8 nmol x h(-1) x g(-1)), less in the red gastrocnemius (117 +/- 8.3), and least in the white gastrocnemius (97 +/- 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest Cr(total) and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated.     

Formation of products of anaerobic metabolism in the ischemic myocardium

The effect of ischemia on the formation of products of anaerobic metabolism and their release into the cardiac effluent in isolated perfused guinea pig hearts was studied. During 30 min normothermal ischemia, the myocardial ATP and phosphocreatine levels decreased to 34% and 15% of the initial values, respectively. The net alanine formation in ischemia was approximately a stoichiometric glutamate decrease; the increase in the tissue malate content corresponded to the aspartate----oxaloacetate----malate anaplerotic flux, the succinate production being commensurable to alpha-ketoglutaric acid formation in the alanine aminotransferase reaction. Using 1H-NMR, it was shown that the release of trace amounts of lactate, alanine, succinate, creatine and pyruvate into cardiac effluents occurred during the first 5 minutes of reperfusion. The rate of metabolite release decreased in the following order: lactate much greater than alanine greater than succinate greater than creatine. By the 30th minute of reperfusion, the decrease in the tissue levels of these metabolites to preischemic values was accompanied by the recovery of ATP and phosphocreatine to 65% and 90% of the initial levels, respectively. The data obtained suggest that the formation and release of alanine, creatine or succinate as well as lactate from ischemic myocardium may testify to significant disturbances in energy metabolism of the myocardium.     

Is the damaging action of catecholamines on the myocardium realized via hyperstimulation of the beta receptors?

Experiments on an isolated rat heart were made to compare the damaging action on the myocardium of catecholamines (noradrenaline, adrenaline and isoproterenol) differing in the affinity for beta-receptors. The damage to myocardial cells was evaluated from the release into the perfusate of intracellular enzymes (creatine phosphokinase and lactate dehydrogenase) and the number of contracture damaged myocytes. Noradrenaline exerted the most powerful damaging action on the myocardium at a concentration of 10(-6) M. Perfusion of the heart with isoproterenol at concentrations of 10(-6) M and 10(-5) M did not lead to the affection of cardiomyocytes. It was isoproterenol concentration exceeding noradrenaline concentration 100 times that produced an increase in the rate of the release of the enzymes to the perfusate and a rise of the number of contractures in the myocardium, with the above increase being less than that provoked by adrenaline and noradrenaline (10(-6) M). It is concluded that the mechanism of the cardiotoxic effect of catecholamines cannot be reduced only to their effect on myocardial beta-receptors.     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

Ability of a phosphocreatine-myofibrillar creatine kinase system to prevent the rigor tension of myocardial fibers

In the calcium-free medium the EGTA-treated rat myocardial fibres developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and the maximal tension at 0.1 mM. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. In the presence of MgADP phosphocreatine decreased rigor tension more rapidly and to the higher extent than MgATP. At 5 mM MgADP half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the km value for phosphocreatine in the creatine kinase reaction. These results demonstrate that the native creatine kinase in the EGTA-treated fibres is able to create high local ATP concentration in the myofibrillar compartment at the expense of phosphocreatine under the conditions of deficiency or even absence of ATP. It appears that at the energy supply disturbances the myocardial contracture develops at least partially due to low activity of the myofibrillar creatine kinase because of phosphocreatine deficiency.     

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.     

A decrease in cardiac sensitivity after ischemia to a change in the extracellular concentration of calcium ions]

Reperfusion of the heart 30 min. after ischemia causes slight recovery of contractility and content of macroergic compounds in the myocardium tissue. Recovery of perfusion by the hypercalcium medium (0.05 mol/l) improves metabolism of the myocardium 30 min after ischemia. However, further perfusion by solution with physiological content of Ca2+ is followed by the development of the myocardium contracture, essential decrease in extracellular concentration of ATP and phosphocreatine. An increase in the extracellular sodium concentration and addition of macroergic compounds (ATR, phosphocreatine) adenosine, when reperfusing the heart by hypocalcium solution, improve the postischemic state of the myocardium and protect it from injuries during the following recovery of physiological Ca2+ content in the extracellular medium.     

ATP,creatine phosphate,and mechanical activity in rainbow trout myocardium under inhibition of glycolysis and cell respiration

Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N₂. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO₂. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted.     

Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes

Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]e), were further decreased in the presence of 10 mM [Na+]e), to 51.4 +/- 4.0 and 6.80 +/- 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 +/- 0.10 mM to 0.66 +/- 0.13 mM (p less than 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N-methyl-D-glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N-acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

Relation between the energy state of the myocardium and the release of products of anaerobic metabolism

The relationships between cellular energy parameters and succinate, alanine and creatine release from isolated guinea pig hearts were studied during a 50 min perfusion (0.2 ml/min) with 5.5 mM glucose or 5 mM sodium acetate. Compared to glucose-perfused hearts, a more rapid ATP depletion accompanied by an increased succinate and creatine release was observed during underperfusion with acetate. The succinate and alanine accumulation in the myocardial effluent was related to a decrease in tissue ATP; the creatine release showed a close inverse correlation with the tissue phosphocreatine/creatine ratio. Hyperbolic and linear relationships were found between these indices for glucose- and acetate-perfused hearts, respectively. The logarithm of tissue ATP had negative linear correlations with the perfusate succinate/creatine ratio for the both substrates. The experimental results suggest that succinate, creatine and alanine assays in the myocardial effluent may be used for the assessment of the energy state of ischemic heart.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Effects of ammonium on energy metabolism and intracellular pH in guinea pig cerebral cortex studied by P and H nuclear magnetic resonance spectroscopy

³¹P and ¹H nuclear magnetic resonance spectroscopy were used to study the effects of ammonium on high-energy phosphates, intracellular pH and lactate in guinea pig cerebral cortex in vitro. In the presence of glucose, 1 mM ammonium caused an intracellular acidification by 0.2–0.3 pH units without a change in phosphocreatine/ATP (PCr/ATP) ratio, lactate concentration or oxygen uptake. At concentrations of 5 mM or greater, NH₄⁺ caused an energy failure and an increase in tissue lactate, together with a drop in intracellular pH. A split in the inorganic phosphate resonance was observed during the exposure to both 20 mM NH₄⁺ and 20 mM K⁺ indicating heterogeneity of the volume-averaged intracellular pH. Cortical brain slices incubated in the presence of 10 mM lactate maintained PCr/ATP ratio and intracellular pH at similar levels as in the presence of glucose, but 1 mM NH₄⁺ caused a fall in PCr/ATP. Both 20 mM NH₄⁺ and 20 mM K⁺ stimulated oxygen uptake of the preparation with glucose or lactate as substrate. These results show that the only acute effect of 1 mM NH₄⁺ in the presence of glucose is an intracellular acidification whereas energetic consequences develop at high levels of this neurotoxic agent.     

The metabolic state of muscle in the isolated perfused rat hemicorpus in relation to rates of protein synthesis.

Measures of perfusion adequacy in perfused rat hemicorpus preparations were investigated as potential indices of tissue function during studies of muscle protein metabolism. Perfusion under normal conditions for up to 80 min resulted in rates of protein synthesis and concentrations of ATP in muscle that were similar to those in vivo, but phosphocreatine in muscle gradually decreased and muscle lactate increased. Hypoxic conditions led to lower rates of protein synthesis, lower phospho-creatine and raised lactate contents in muscle compared with normal perfusions, and ATP was slightly decreased. Hypoxic preparations also released more lactate and K+ into the medium and had higher perfusion pressures, but glucose uptake and muscle water content were not altered. In totally ischaemic muscle, concentrations of ATP and phosphocreatine were even lower than in hypoxic muscle, and that of lactate was higher. From 11 preparations perfused for 60 min under normal conditions, three were selected on the basis of lower muscle ATP content than the others. Preparations with low ATP also showed lower muscle phosphocreatine concentrations, O2 uptake and CO2 output, as well as higher perfusion pressure and muscle lactate concentrations than in the remaining preparations, but muscle water, ADP and AMP concentrations and lactate and K+ flux were no different. In perfusions extended to 3 h, deterioration of function was more apparent. There were significant correlations between rates of protein synthesis and the concentrations of ATP, phosphocreatine and lactate in two different muscles (r = 0.756-0.929), but not with any of the other indices investigated. Taken overall, these experiments showed that concentrations of ADP, AMP and water in muscle, rates of lactate and glucose metabolism, K+ output, perfusion pressure and blood gas parameters were unsuitable for distinguishing unsound from sound preparations, because they did not consistently demonstrate differences, or could not be ascribed to only muscle metabolism. It was found that ATP, phosphocreatine and lactate concentrations in muscle were the best indicators of impaired metabolic state in studies of protein synthesis. Measurements of these could be used on a routine basis for rejecting unsatisfactory preparations.     

31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices.

The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.     

Effect of phosphocreatine on the lysophosphoglyceride levels in total ischemia of the rat myocardium

The phospholipid composition of the crude plasma membrane fraction of Langendorff perfused rat hearts has been studied. The effect of phosphocreatine (PCr) and 3-phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI) on lysophosphoglycerides (LPG) level in this fraction isolated from hearts that were totally ischemic for 8 minutes, has been examined. The absolute and relative contents of LPG were significantly increased in ischemic hearts: the lysophosphatidylcholine content was elevated by 94% and that of lysophosphatidylethanolamine--by 77%. Accumulation of these LPG in ischemic myocardium was completely inhibited in the presence of 10 mM PCr or PIMOI in the perfusate. LPG may play a key role in the destruction of sarcolemma. Therefore, these data allow to assume that the protective effect of PCr and PIMOI on the sarcolemma of ischemic myocardium may be the result of their influence on the phospholipid metabolism in the ischemic region of the heart.     

Changes of tissue creatine concentrations upon oral supplementation of creatine-monohydrate in various animal species

Creatine is a nutritional supplement with major application as ergogenic and neuroprotective substrate. Varying supplementation protocols differing in dosage and duration have been applied but systematic studies of total creatine (creatine and phosphocreatine) content in the various organs of interest are lacking. We investigated changes of total creatine concentrations in brain, muscle, heart, kidney, liver, lung and venous/portal plasma of guinea pigs, mice and rats in response to 2-8 weeks oral creatine-monohydrate supplementation (1.3-2 g/kg/d; 1.4-2.8% of dietary intake). Analysis of creatine and phosphocreatine content was performed by high performance liquid chromatography. Total creatine was determined as the sum of creatine and phosphocreatine. Presupplementation total creatine concentrations were high in brain, skeletal and heart muscle (10-22 micromol/g wet weight), and low in liver, kidney and lung (5-8 micromol/g wet weight). During creatine supplementation, the relative increase of total creatine was low (15-55% of presupplementation values) in organs with high presupplementation concentrations, and high (260-500% of presupplementation values) in organs with low presupplementation concentrations. The increase of total creatine concentrations was most pronounced after 4 weeks of supplementation. In muscle, brain, kidney and lungs, an additional increase (p<0.01) was observed between 2-4 and 2-8 weeks of supplementation. Absolute concentrations of phosphocreatine increased, but there was no increase of the relative (percentual) proportion of phosphocreatine (14-45%) during supplementation. Statistical comparison of total creatine concentrations across the species revealed no systematically differences in organ distribution and in time points of supplementation. Results suggest that in organs with low presupplementation creatine levels (liver, kidney), a major determinant of creatine uptake is an extra-intracellular concentration gradient. In organs with high presupplementation total creatine levels like brain, skeletal and heart muscle, the maximum capacity of creatine accumulation is low compared to other organs. A supplementation period of 2 to 4 weeks is necessary for significant augmentation of the creatine pool in these organs.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.