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Biomedical subjects

G K Radda

Publications and source records attributed to G K Radda.

At least 289 records · Page 16Linked to original sources
PubMed1985

Creatine kinase kinetics, ATP turnover, and cardiac performance in hearts depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed a diet containing 1% of the creatine substrate analogue beta-guanidinopropionic acid for 6-10 weeks. 31P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 mumol/g dry wt in guanidinopropionic acid-fed animals but no change in [Pi], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine----ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 mumol/g dry wt per s in control hearts and decreased 4-fold to 2.5-2.8 mumol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2-7.8 mumol/g dry per s. These rates are 1.5-3-times greater than the rate of the phosphocreatine----ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.

Adenosine Triphosphate↗
PubMed1985

A comparison of in vivo catalysis by creatine kinase in avian skeletal muscles with different fibre composition.

The maximum activity of creatine kinase in vitro is similar in the pectoralis major muscle of the chicken and the duck. However, the flux (phosphocreatine to ATP) as measured by 31P saturation transfer NMR in vivo is almost 2-fold higher in the duck. This apparent discrepancy can be accounted for by the differences in the cytosolic free ADP concentrations in resting muscle.

Adenosine Diphosphate↗
PubMed1985

Measurements of exchange in the reaction catalysed by creatine kinase using 14C and 15N isotope labels and the NMR technique of saturation transfer.

31P-NMR measurements of saturation transfer have been used to measure exchange between the gamma-phosphate of ATP and phosphocreatine and between the beta-phosphate of ATP and the beta-phosphate of ADP in the reaction catalysed by creatine kinase in vitro. The calculated exchange fluxes have been compared with measurements of 15N label exchange between creatine and phosphocreatine and 14C label exchange between ATP and ADP. At pH 8.0 the fluxes between phosphocreatine and the gamma-phosphate of ATP and between the beta-phosphates of ATP and ADP, measured by saturation transfer, were the same and equal, within experimental error, to the fluxes between creatine and phosphocreatine, measured by 15N label exchange, and between ADP and ATP, measured by 14C label exchange. At pH 7.0 the flux between phosphocreatine and the gamma-phosphate of ATP, measured by saturation transfer, was equal, within experimental error, to the flux between creatine and phosphocreatine, measured by 15N label exchange. However, at low ADP concentrations (less than 0.2 mM), the flux between ATP and ADP measured by saturation transfer was significantly less than that between phosphocreatine and ATP and, more importantly, less than the ADP-ATP exchange flux measured by 14C label exchange. The saturation transfer and isotope exchange measurements at pH 7.0 have shown that it is valid to equate saturation transfer measurements of exchange between phosphocreatine and ATP in vivo with the potential for net chemical flux through the reaction. The observed discrepancy at pH 7.0 between the 14C and saturation transfer measurements of ATP----ADP exchange can be explained if there is significant loss of saturation in an intermediate in the exchange reaction. Under these conditions analysis of the exchange according to two-site exchange model is invalid. In magnetisation transfer measurements of exchange in other enzyme catalysed reactions, the possible presence of a kinetically significant intermediate and therefore the validity of data analysis using a two-site exchange model should be considered.

Adenosine Diphosphate↗
PubMed1985

Declining catecholamine secretion in adrenal medulla on prolonged stimulation with acetylcholine.

Perfused pig adrenal glands and cortex-free ox adrenal medullae were stimulated by continuous infusion of 10(-4) M acetylcholine (ACh). Secretion of adrenaline rose to a maximum in approximately 5 min but, after a further 15 min, declined to 36 +/- 19% (+/- S.D.) of maximum for pig (N = 5) and 27 +/- 10% of maximum for ox (N = 3), in spite of continued infusion of ACh. After 20 min, no further significant decline was detectable. Nevertheless, in ox medullae, oxygen consumption measured after stimulation showed no significant change relative to the pre-stimulation value, indicating that the decline in secretion did not arise from a failure of oxidative energy metabolism. In 4 pig adrenal glands subjected to a 1 hr infusion of ACh, adrenaline secreted in the last 20 min was only 52 +/- 10% of that secreted in the first 20 min but, after a 2 hr rest interval, recovered to 74 +/- 18% (P less than 0.05) in the first 20 min of a second 1 hr stimulation. In the same glands, no reproducible recovery was detectable for noradrenaline and, by the last 20 min of the second 1 hr stimulation, noradrenaline secretion had declined to 36 +/- 20% of the initial value, even though only 15% of the noradrenaline originally in the gland had been secreted. It is concluded that, while decline and recovery of adrenaline secretion may, in part, have arisen from desensitization and resensitization of the ACh receptor; the decline in noradrenaline secretion arose mainly from depletion of a readily secreted pool which was considerably smaller than the total in the gland.

Acetylcholine↗
PubMed1985

Enhanced oxygen consumption in adrenal medulla on stimulation with acetylcholine.

When perfused cortex-free ox adrenal medulla was stimulated to secrete catecholamine by infusion of 0.1 mM acetylcholine for 4 min, the oxygen consumption increased to a value which was 0.15 +/- 0.07 mumole O2/min/g wet weight (+/- S.D., N = 12) above the pre-stimulation value of 0.49 +/- 0.15 (P less than 0.001). 1.4 +/- 0.9 (+/- S.D., N = 12) mole of catecholamine was secreted per mole of enhanced O2 consumption in the 16 min following the start of the stimulation. The rate of ATP hydrolysis by the proton-translocating Mg-ATPase of the chromaffin granule may increase on fusing with the plasma membrane of the chromaffin cell during exocytosis. However, from the amount of catecholamine secreted, this was estimated to account for less than 17% of the oxygen consumption increase. The amount of catecholamine secreted by 4 min 0.1 mM acetylcholine stimulations correlated with the enhancement of oxygen consumption (r = 0.82, P less than 0.001) but, on stimulation with 60 microM veratridine for 4 min, O2 consumption enhancement was anomalously low. This dependence on mode of stimulation suggests that ATP consumption in exocytosis itself is an inadequate explanation. Ouabain-sensitive oxygen consumption rose from undetectable levels to 18 +/- 8% (+/- S.D., N = 4) of the basal respiration during prolonged 0.1 mM acetylcholine stimulation in the absence of Ca, indicating that Na,K-ATPase was not responsible for all of the oxygen consumption enhancement.

Acetylcholine↗
PubMed1985

Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy.

Abnormal mitochondria are an increasingly recognized cause of neuromuscular disease. We have used phosphorus magnetic resonance spectroscopy to monitor noninvasively the metabolism of high-energy phosphates and the intracellular pH of human skeletal muscle in vivo in 12 patients with mitochondrial myopathy. At rest, an abnormality could be demonstrated in 11 of 12 patients. Ten patients had evidence of a reduced muscle energy state with at least one of the following abnormalities: low phosphorylation potential, low phosphocreatine concentration, high adenosine diphosphate concentration, or high inorganic phosphate concentration. Two patients had abnormal resting muscle intracellular pH. In some patients phosphocreatine concentration decreased to low values during exercise despite limited work output. This was not accompanied by particularly severe intracellular acidosis. Evidence of impaired rephosphorylation of adenosine diphosphate to adenosine triphosphate during recovery from exercise was found in approximately half the patients. Phosphorus magnetic resonance spectroscopy is useful in the noninvasive diagnosis of mitochondrial myopathies and in defining the pathophysiological basis of these disorders.

Adenosine Diphosphate↗
PubMed1985

A method for localizing high-resolution NMR spectra from human subjects.

Several methods are available for the localization of high-resolution spectra. When the sample is a human subject, it may be difficult to realize a viable experimental protocol because the size and geometry of the subject often conflict with efficient probe design. In this paper a spectroscopic localization method is described which is based on the method of rotating frame imaging. A novel surface-coil probe has been developed so that this experiment can be performed on a large sample. 31P spectra from the thorax of a human subject are presented in which signals from liver and intercostal muscle are resolved. These data demonstrate that spatially resolved spectra of metabolites in vivo may be obtained in a time that is acceptable for patient examination.

Humans↗
PubMed1985

A mitochondrial encephalomyopathy. A combined 31P magnetic resonance and biochemical investigation.

A 15-year-old girl presented with recurrent encephalopathic episodes, epilepsy, myopathy and chronic lactic acidosis. A muscle biopsy revealed the presence of ragged red fibres and mitochondria with paracrystalline inclusions. Biochemical studies on freshly isolated skeletal muscle mitochondria demonstrated a deficiency of NADH-CoQ reductase activity. Investigation of her gastrocnemius muscle at rest by phosphorus nuclear magnetic resonance displayed a reduced phosphocreatine concentration with elevated levels of inorganic phosphate and ADP. Abnormalities were also apparent in her brain spectrum. It is therefore possible that the mitochondrial defect present in skeletal muscle is also being expressed in the brain.

Adolescent↗
PubMed1985

A phosphorus-31 nuclear magnetic resonance study of effects of altered thyroid state on cardiac bioenergetics.

We have used 31P nuclear magnetic resonance (NMR) techniques to characterize bioenergetic changes in the Langendorff-perfused rat heart accompanying alterations in thyroid state. Cytosolic phosphocreatine and inorganic phosphate concentrations changed significantly in both the hypo- and hyperthyroid groups compared to controls; the calculated phosphorylation potential [( ATP]/[ADP][Pi]) increased by 60% in hypothyroidism and decreased by 60% in hyperthyroidism relative to the euthyroid value of 47 X 10(3) M-1. Creatine phosphokinase (CPK) and mitochondrial ATP synthase rates were measured in the intact tissue using a saturation-transfer NMR method. There were no significant differences in the measured fluxes through the CPK reaction among the three groups (4.24 +/- 1.00 mM X sec-1 for the euthyroid group). Although O2 consumption increased by 46% in hearts from hyperthyroid animals, no change in the measured mitochondrial ATP synthase flux was observed compared to the euthyroid flux of 1.05 +/- 0.11 mM X sec-1. These results suggest that the apparent in situ P/O ratio of mitochondria in hearts from hyperthyroid animals is reduced relative to that in euthyroid controls.

ATP Synthetase Complexes↗
PubMed1985

In vivo phosphorus-31 nuclear magnetic resonance study of the regional metabolic response to cardiac ischemia.

We applied 31P nuclear magnetic resonance to the study of acute ischemia in vivo. In open-chest rabbits, the left anterior descending coronary artery was occluded after control spectra were obtained. Phosphocreatine concentration decreased in the first minute and ATP concentration declined subsequently, and pH decreased to 5.8 within 20 min. Intracellular acidosis was greater than that previously reported for the isolated perfused rabbit heart.

Acid-Base Equilibrium↗
PubMed1984

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle.

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.

Animals↗
PubMed1984

A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue beta-guanidinopropionic acid.

Rats were fed a diet containing 1% beta-guanidinopropionic acid (GPA) for 6-10 weeks to deplete their skeletal muscle of creatine. 31P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 mumol X g-1) and the [ATP] to 50% (3.3 mumol X g-1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 mumol X g-1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM X s-1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM X s-1). The rate of PGPA resynthesis during recovery (0.018 mM X s-1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM X s-1). The creatine kinase flux (creatine phosphate----ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM X s-1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.

Animals↗
PubMed1984

Excessive intracellular acidosis of skeletal muscle on exercise in a patient with a post-viral exhaustion/fatigue syndrome. A 31P nuclear magnetic resonance study.

A patient with prolonged post-viral exhaustion and excessive fatigue was examined by 31P nuclear magnetic resonance. During exercise, muscles of the forearm demonstrated abnormally early intracellular acidosis for the exercise performed. This was out of proportion to the associated changes in high-energy phosphates. This may represent excessive lactic acid formation resulting from a disorder of metabolic regulation. The metabolic abnormality in this patient could not have been demonstrated by traditional diagnostic techniques.

Adenosine Diphosphate↗
PubMed1984

A comparison of 31P-NMR saturation transfer and isotope-exchange measurements of creatine kinase kinetics in vitro.

31P-NMR measurements of saturation transfer have been used to measure the exchange of phosphate between phosphocreatine and the gamma-phosphate of ATP in the reaction catalysed by creatine kinase in vitro. The similarity of the calculated exchange flux with the flux estimated from an isotope-exchange experiment, in which exchange of 15N label between creatine and phosphocreatine was measured, showed that the two-site-exchange model, normally used in the analysis of saturation-transfer data, is valid in this case. 15N label exchange was monitored using a heteronuclear 31P/15N spin-echo NMR experiment in which the incorporation of 15N label into phosphocreatine was detected by following the phase modulation of the spin-spin coupled 31P resonance. The isotope-exchange experiment should prove to be useful in studies of creatine kinase in systems where the low concentration of the enzyme precludes saturation-transfer measurements, for example in muscle mitochondria preparations.

Creatine Kinase↗
PubMed1984

Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR.

It has been suggested that the rate of phosphocreatine resynthesis after exercise is an index of mitochondrial oxidative metabolism in intact muscle. To investigate this hypothesis, the time courses of metabolite recovery following mild and more severe dynamic exercise of human forearm muscle were compared by means of 31P NMR. Severe exercise resulted in greater net hydrolysis of phosphocreatine and greater intracellular acidosis than light exercise. The rate of phosphocreatine resynthesis was significantly slower during recovery from the more severe exercise. To explain this it was noted that, as a consequence of the high activity of creatine kinase in the sarcoplasm, the [phosphocreatine] at any time is a function of the intracellular pH. Calculations demonstrate that the difference between rates of phosphocreatine recovery after the two exercise protocols was primarily determined by the rates of recovery of the intracellular pH to normal rest values. It is concluded that the calculated rate of recovery of the cytosolic free [ADP] to its pre-exercise concentration may provide a more specific measure of mitochondrial oxidative activity.

Humans↗
PubMed1984

Isolation of a membrane protein by chromatofocusing: cytochrome b-561 of the adrenal chromaffin granule.

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.

Chromaffin Granules↗