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The gracilis muscle was excised from cold-acclimated rats, placed in vitro, and simultaneously perfused via its artery by high pO2 medium and superfused by low pO2 medium. With a doubling of the perfusion rate (from 50 to 100 microliters/min) phosphocreatine and ATP increased by 39% and 44%, respectively.
The purpose of this study was to find out whether Ca2+ is necessary for the protective effect of phosphocreatine (PCr) on ischemic myocardium. Isolated Langendorff-perfused rat hearts were used in the study. When ischemic arrest was induced in Ca(2+)-free buffer, PCr did not exert a protective effect on ischemic myocardium. PCr improved postischemic contractile recovery of hearts subjected to ischemia in perfusion media containing 0.5 and 1.2 mmol/l Ca2+. Phosphoarginine, a structural analogue of PCr which possesses Ca(2+)-binding property similar to that of PCr did not exert any protective effect on ischemic myocardium. The effects of PCr and Ca2+ on lipid order of sarcolemmal vesicles from canine heart were studied by using ESR spectroscopy. PCr made membrane phospholipids more tightly packed at mildly acidic and neutral pH, but did not at pH 8.5. Although Ca2+ itself did not influence the membrane structure at pH 5.5, it potentiated the effect of PCr on sarcolemmal phospholipids. Thus, the protective effect of PCr on ischemic myocardium is not attributed to its Ca2+ binding properly, but Ca2+ is a necessary component of the mechanism of protective effect of PCr on ischemic myocardium.
We measured ATP, phosphocreatine (PCr), inorganic phosphate (Pi), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O2 deliveries using 31P nuclear magnetic resonance spectroscopy (31P NMR) to evaluate changes in energy metabolism in relation to O2 availability. Delivery of O2 to muscles was altered by controlling the fractional concentration of inspired oxygen (F1O2) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr: (PCr + Pi) during exercise decreased as a function of F1O2 even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08 F1O2. Change in the PCr: (PCr + Pi) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O2 delivery at 0.50 F1O2, but was significantly limited at 0.21 F1O2 or below. Change in the intracellular pH at 0.08 F1O2 could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr: (PCr + Pi) ratio and the intracellular pH revealed the existence of a critical PCr: (PCr + Pi) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.
Eight healthy untrained male volunteers pedalled a cycle ergometer according to two exercise protocols: the first involved step-wise increasing physical exercise to maximal (MPE); the second involved prolonged (35 min) submaximal physical exercise (PPE) at 70% of the individual's maximal oxygen uptake. Each volunteer performed these exercise twice, following either an intravenous injection of phosphocreatine (PCr) or a placebo of an isotonic NaCl solution. Anaerobic threshold (AT) was determined from the point of departure of the ventilatory response from linearity and from the sudden increase in venous blood lactate concentrations during MPE. After exercise following placebo administration we observed increases in concentrations of blood substrates, plasma adrenocorticotropin (ACTH), growth hormone and cortisol and in the number of glucocorticoid receptors in lymphocytes without changes in the dissociation constant. Intravenous administration of PCr (starting 1 day before exercise) led to an increase in the total workload (on average by 5.8%) and in AT (on average by 6.8%) during MPE and to a better tolerance of exercise during PPE. Following PCr administration we observed lower blood lactate concentrations and different patterns of some enzyme activities, less pronounced changes in plasma ACTH and cortisol concentrations and in glucocorticoid binding in lymphocytes, but no changes in plasma growth hormone concentrations compared to the placebo. The results showed that intense physical exercise led not only to increases in blood hormone concentrations but also to an increase in the density of glucocorticoid receptors in lymphocytes. Intravenous PCr injection led to smaller changes in ACTH and cortisol concentrations as well as to a lower activation of glucocorticoid binding in lymphocytes.
31P saturation-transfer nuclear magnetic resonance spectroscopy was used to study skeletal muscle phosphocreatine (PCr) flux in healthy male volunteers. Data analysis included consideration of effects from incomplete saturation and radiofrequency spillover. Spectra were recorded from the resting gastrocnemius muscle before and after 6 days of creatine monohydrate (Cr-H2O) intake (20 g/day). Parallel to an improved muscle performance during maximal intermittent exercise following Cr-H2O supplementation, the concentration of PCr increased (P=0.01) by 23% (34.9+/-2.8 mmol/l vs. 28.6+/-2.7 mmol/l), whereas other metabolites were unaffected (inorganic phosphate: 4.3+/-1.4 mmol/l, free intracellular Mg(2+): 1.1+/-0.7 mmol/l, cytosolic pH: 7.04+/-0.02). Forward and reverse fluxes through the creatine kinase (CK) reaction did not change significantly from their baseline levels (v(for): 11.8+/-5.4 mmol/l per second vs. 15.3+/-6.8 mmol/l per second, (v(rev): 9.5+/-3.4 mmol/l per second vs. 10.9+/-3.7 mmol/l per second). The rate of PCr resynthesis in resting muscle is not limited by the CK reaction, which is near equilibrium. Consequently, the post-load increase in total creatine has no effect on the unidirectional CK reaction rates.
L-Glutamate, a major excitatory amino acid, plays an important role in learning and memory. L-Glutamate uptake into synaptic vesicles is an ATP-dependent process. Exposure of neurons to high, sustained extracellular concentrations of glutamate results in excitotoxicity. Elevated levels of phosphomonoesters (PMEs), phosphodiesters (PDEs), and phosphocreatine (PCr) have been reported in Alzheimer disease (AD). In this article, the effects of selected PMEs, PDEs, and PCr on vesicular L-[3H]glutamate uptake into isolated bovine synaptic vesicles are investigated. D-myo-Inositol-1-monophosphate (I1P), D-myo-inositol-2-monophosphate (I2P), sn-glycero-3-phosphate, (alpha-GP) and PCr significantly stimulated L-[3H]glutamate uptake into synaptic vesicles. Phosphoethanolamine (PE), phosphocholine (PC), L-phosphoserine (L-PS) sn-glycero-3-phosphocholine (GPC), and sn-glycero-3-phosphoethanolamine (GPE) had little or no effect on vesicular L-glutamate uptake. These observations suggested that the vesicular uptake of glutamate can be regulated by endogenous PMEs and PCr. The mechanism of activation by I1P, I2P, and alpha-GP appears to be stimulation of Mg(2+)-ATPase activity. These effects on vesicular glutamate uptake may be important in diseases in which the levels of these metabolites are altered, as they are in AD.
Our aim was to develop an exercise protocol using 31P-magnetic resonance spectroscopy (31P-MRS), which can discriminate between malignant hyperthermia-susceptible (MHS) individuals and controls. MRS spectra of the forearm muscles were recorded at rest, during and after a standardized exercise protocol in 10 MHS patients and compared with spectra obtained in 10 controls. There was no difference in resting intracellular pH (pHi) or PCr/(Pi+PCr) ratio between the groups (PCr = phosphocreatine, Pi = inorganic phosphorus). At the end of the exercise and during the initial recovery phase, the pHi and PCr/(Pi+PCr) ratio were significantly lower in the MHS group ([pHi: 6.37 (0.07) for MHS vs 6.70 (0.05) for controls, P < 0.005; PCr/(Pi+PCr): 0.784 (0.017) for MHS vs 0.954 (0.020) for controls, P < 0.0005]). For PCr/(Pi+PCr), complete separation between the two groups was observed during the initial recovery phase. The mean recovery time of PCr/(Pi+PCr) was 0.57 min for the control group and 1.28 min for the MHS group. The slower recovery of PCr/(Pi+PCr) is likely to be caused by a combination of several factors, including the lower pHi in MHS subjects at the start of recovery (inhibiting ATP production) and excessive sarcoplasmic calcium overload (causing continued enzyme activation and ATP consumption). Our exercise protocol can be a valuable adjunct to discriminate between MHS and non susceptible subjects.
Commonly, muscle phosphocreatine (PCr) recovery from exercise has been fitted to a monoexponential function. However, a number of experiments indicate that at low muscle pH, a monoexponential fit is not suitable. We have performed in vivo 31P-MRS measurements of PCr during recovery from high-intensity intermittent exercise where muscle pH dropped below 6.5 (35 out of 40 cases). Results of a statistical analysis showed that monoexponentiality should be rejected in 32 out of 40 cases. Therefore, a Monte Carlo simulation was carried out to test the quality of competing models used in the literature at low pH: monoexponential, biexponential and changing rate utilization resource (CRUR). For each model, random Gaussian-distributed errors were imposed on simulated PCr recovery before performing the fits. A monoexponential function (three estimated parameters) provided a correct estimation of parameters (unbiased, normally distributed, poorly correlated estimates) and, therefore, should be preferred. When alternative functions are required, as in 32 cases out of 40 in the present study, it is demonstrated that a biexponential function (five estimated parameters) is not well suited (estimates were correlated), whereas a CRUR function (four estimated parameters) provides correct estimation of the parameters. It is concluded that a biexponential fit to PCr recovery is too sensitive to experimental errors to be practicable. Statistical and physiological relevance of CRUR are discussed.
ATP and phosphocreatine (PCr) were measured in randomly selected single fibres from control, 1- and 8-day low-frequency-stimulated rabbit tibialis anterior muscles. The fibres were classified according to their myosin heavy chain (MHC) complement as type I, IIA or IID. In 1-day stimulated muscle, which has previously been shown to exhibit a steep decline in force output, two fibre populations could be distinguished according to either normal or markedly depressed PCr levels. The fibre population exhibiting normal PCr levels encompassed a major fraction (65%) of type IID fibres and a minor fraction (35%) of IIA fibres. The population with reduced PCr levels comprised type I fibres (@50% reduced), the majority of type IIA fibres (@80% reduced), and a minor fraction of type IID fibres (@70% reduced). Levels of ATP were unaltered in type I and IIA fibres, but were @ 20% reduced in those IID fibres that exhibited low PCr levels. Assuming that those fibres that displayed reduced PCr levels were contracting, the IID and IIA fibres with normal PCr levels were regarded as metabolically recovering, non-contracting fibres. As previously shown, these fibres are transiently refractory during the early phase of low-frequency stimulation. After 8 days of chronic low-frequency stimulation, when force was shown to rise again, most fibres appeared more uniform with regard to reduced PCr and ATP levels. Our results suggest that PCr can be used as a sensitive measure of the degree of activity in single-fibre studies.
The recovery of high-energy phosphate levels in single human skeletal muscle fibres following short-term maximal (all-out) exercise was investigated. Three male volunteers exercised maximally for 25 s on an isokinetic cycling ergometer. Muscle biopsy samples from the vastus lateralis were collected at rest, immediately post-exercise and at 1.5 min of recovery. The subjects also performed a second exercise bout 1.5 min after the first, on a separate occasion. Single muscle fibres were dissected, characterized and assigned to one of four groups according to their myosin heavy chain (MyHC) isoform content; namely, type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fibres were analysed for adenosine 5'-triphosphate (ATP), inosine-5'-monophosphate (IMP), phosphocreatine (PCr) and creatine (Cr) levels. Type I fibres had a lower Cr content than type II fibres (P<0.01). Within type II fibres resting [PCr] increased with increasing MyHC IIX isoform content (r=0.59, P<0.01). Post-exercise [PCr] was very low in all fibre groups (P<0.01 versus rest) while great reductions in ATP were also observed (P<0.01 versus rest), especially in the type II fibre groups. [PCr] at 1.5 min of recovery was still lower compared to rest for all fibre groups (P<0.01) especially in the IIAx and IIXa fibres.
AIM: The aim of this study was to determine the "spin-lattice relaxation" or "T1" time constant of phosphocreatine in the human gastrocnemius muscle, both at rest and during aerobic exercise, when a clinical nuclear magnetic resonance (NMR) scanner is available. MATERIALS AND METHODS: Using the multipoint saturation recovery technique, we tested four acquisition protocols differing in number and duration of repetition times. Moreover, two mathematical models describing the phenomenon were also evaluated. Protocols and models were tested on a phantom containing 1 kg of distilled water in which were dissolved 5.88 g sodium chloride, 1 cm3 phosphoric acid and 7.58 g sodium phosphate dodecahydrate. One protocol only was used on a group of four healthy volunteers both at rest and during exercise. Each volunteer repeated the rest-exercise sequence three times. RESULTS AND CONCLUSIONS: On the phantom, the average T1 values for the various protocols and mathematical models used proved to differ widely, ranging from 0.61 s to 7.20 s. On volunteers, the T1 values obtained at rest and during exercise were not significantly different--0.91 s on average. Correction of this T1 value with the results obtained using the phantom provides a T1 value of 5.73 s, which is comparable with the value reported in the literature for resting conditions only.
The cellular electrophysiologic effects of exogenous phosphocreatine (PCr) were analyzed to ascertain its purported antiarrhythmic properties during myocardial ischemia and reperfusion. Transmembrane potentials were recorded from isolated guinea pig papillary muscles and Purkinje fibers studied in vitro. Under control, normoxic conditions, 10 mmol/L PCr significantly increased the action potential duration (measured at 90% of repolarization) in ventricular muscle by 14.6 +/- 3.3 msec and the effective refractory period by 11.5 +/- 3.8 msec (both p less than 0.01). Under ischemic-like conditions (hypoxia, lactic acidosis, elevated [K+]o, zero substrate) PCr had no effect. Phosphocreatinine, a related compound that is not a direct substrate in the creatine kinase reaction, acted similarly to PCr suggesting that alterations induced by PCr did not involve a change in the energy state of cells. However, PCr reduced free [Ca2+]o by nearly 20%, and its electrical effects under normoxic conditions could be largely reversed by a concomitant 20% increase in [Ca2+]o. In Purkinje fibers superfused with low [K+]o-Tyrode's solution to elicit conditions of Ca2+ overload, delayed afterdepolarizations and triggered responses were reversibly inhibited by PCr. These data suggest that the antiarrhythmic effects of PCr in situ may involve prolongation of the effective refractory period in nonischemic tissue or attenuation of membrane changes elicited by Ca2+ overload in ischemic cells. The mechanism by which PCr produces these effects may be related in part to changes in extracellular Ca2+ composition.
Flux rates for phosphate metabolites were calculated using the equation for radial diffusion, assuming heart intracellular conditions and a 5% concentration gradient. The data show that while the flux of phosphocreatine is about 3 times faster than ATP, both are more than two orders of magnitude greater than the known maximum rate of ATP utilization. In contrast, since the concentration of free ADP is very low, its flux is below the maximum rate of ATP turnover, while the flux of creatine is almost 3 orders of magnitude greater than ADP. The data suggest that the rate of high-energy phosphate production could be limited by ADP diffusion, with creatine thus substituting as the primary cytoplasmic-mitochondrial phosphate acceptor.
Rates of ATP metabolism generally are higher in cerebral gray matter compared to white matter. In order to study the physiology of this regional difference in vivo, the 1-dimensional chemical shift imaging technique (1D-CSI) was used to acquire 31P nuclear magnetic resonance spectra from 2.5 mm slices of 4-week old piglet brains. Spectra from predominantly gray matter slices (estimated 76% gray matter, 7 mm below the scalp) were compared to predominantly white matter slices (56% estimated white matter, 13 mm below the scalp) as assessed by magnetic resonance images. The 1D-CSI technique introduced no systematic changes in the ratio of signals from a single chamber phantom containing a phosphocreatine (PCr) and ATP solution. Gray matter slices showed a PCr/NTP ratio of 0.93 +/- 0.11 (mean +/- S.D.) using a 2 s interpulse interval, a value very close to the ratio in surface coil localized spectra. The predominantly white matter slices showed a PCr/NTP ratio of 1.32 +/- 0.18 (P < 0.02 for gray versus white matter). Using the estimated percentages of gray and white matter in the two slices and calculated concentrations from fully relaxed spectra, the gray matter PCr/NTP ratio is approximately 0.77, while the ratio in white matter is approximately 2.18. The difference in PCr/NTP measured in vivo suggests that either the total NTP concentration is higher or the steady state PCr concentration is lower in gray matter than in white matter in the piglet brain.
Effects of accumulation of phosphocreatine (P-creatine) on utilization and restoration of high-energy phosphates during anoxia and recovery were studied using thin hippocampal slices from the guinea pig. Incubation of slices with creatine (5 to 30 mM) increased the content of tissue P-creatine, in a linear fashion, and these increases depended on the concentration of creatine and the incubation period to as long as 3 h. Concentration of ATP in the slices was not altered. A high concentration of tissue P-creatine (40 to 100 mmol/kg protein) accumulated by incubation with creatine reduced the rate of ATP exhaustion during anoxia whereas P-creatine itself was rapidly decreased. During reoxygenation after anoxia, concentrations of ATP and P-creatine rapidly recovered in slices preincubated with creatine. Concentrations of P-creatine previously exhausted during anoxia increased to 100 mmol/kg protein after 25 min of reoxygenation whereas the concentration was 35 mmol/kg in the control slice. Elevation of the concentration of P-creatine may therefore prolong the survival time of brain tissue during anoxia and facilitate recovery during reoxygenation.
Phosphocreatine (PCr) is a critical intracellular energy reservoir used in the regeneration of ATP. The aim of this study was to determine the efficacy of exogenously added PCr on preservation of renal function in an in vitro model. The renal artery and ureter of a rat were cannulated and the kidney was subjected to 45 min of normothermic in vivo ischemia. The kidneys were then perfused ex vivo with either a Krebs-bicarbonate solution (Krebs) or a Krebs solution containing 3 mM PCr or an osmotically balanced solution containing 3 mM PCr. Our results indicate that the perfusion of kidneys subjected to 45 min of warm ischemia with solutions containing PCr resulted in significant improvements in GFR, RPF, and V, FRNa and FRH2O compared to KREBS alone. This suggests that the important factor in preservation of kidney function after an initial ischemic insult may be the addition of PCr rather that the electrolyte solution used.
Phosphorus-31 magnetic resonance spectroscopy can be used to study intracellular biochemistry non-invasively by measuring the relative proportions of high energy phosphates. Study of deteriorating cardiac metabolism might be useful in the management of hypertrophy and heart failure. 31P magnetic resonance spectroscopy was carried out in fourteen patients with aortic valve disease (six with aortic stenosis, eight with aortic incompetence). Six patients were receiving treatment for symptoms of heart failure. The phosphocreatine (PCr) to ATP ratio in these patients (1.1 [SD 0.32]) was significantly lower than that in thirteen controls (1.5 [0.2], p less than 0.001) or in the eight patients who did not have symptoms of heart failure (1.56 [0.15], p less than 0.0035). These findings indicate that heart failure in aortic valve disease is associated with low PCr, which could be due to loss of intracellular creatine. The measurement could eventually have a role in helping to determine the optimum timing for aortic valve replacement.
Brain phosphorus metabolism was measured by 31P-MRS in 15 patients with bipolar II disorder (BP II) and 14 patients with bipolar I disorder (BP I). Phosphocreatine (PCr)levels were significantly lower in patients with BP II in all three psychiatric states compared to 59 normal controls (PCr (%) was 13.5 +/- 1.5 (mean +/- SD) for controls, and 12.2 +/- 1.7, 12.1 +/- 1.3, 12.0 +/- 1.9 for hypomanic, euthymic and depressed bipolar II patients respectively). High values of phosphomonoester (PME) were found in BP II patients in the hypomanic and depressive states, but PME values in the euthymic state did not differ significantly from controls. Intracellular pH of BP II patients in all three psychiatric phases was similar to control values, whereas euthymic BP I patients had lower pH values. These results suggest that brain high energy phosphate metabolism may be impaired in BP II and that there may be pathophysiological differences between BP I and BP II.