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Biomedical subjects

G K Radda

Publications and source records attributed to G K Radda.

At least 271 records · Page 15Linked to original sources
PubMed1986

Functional coupling between enzymes of the chromaffin granule membrane.

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.

Animals↗
PubMed1986

Electron transfer across the chromaffin granule membrane. Use of EPR to demonstrate reduction of intravesicular ascorbate radical by the extravesicular mitochondrial NADH:ascorbate radical oxidoreductase.

A two-compartment electron paramagnetic resonance system has been developed in which the membrane-impermeable spin probe Ni(en)2+3 is used to selectively eliminate the EPR signal from extravesicular ascorbate radical, such that radicals in intra- and extravesicular compartments can be distinguished. Using this system, we have shown that an increase in ascorbate radical in the extravesicular medium is reflected by an increase in ascorbate radical within resealed chromaffin granule ghosts containing trapped ascorbate but has no effect on radical concentrations inside liposomes containing ascorbate. This indicates that the chromaffin granule membrane contains a component, not present in liposomes, that allows equilibration between the intra- and extravesicular ascorbate/ascorbate radical couples. This component is probably cytochrome b561. We further show that activation of the mitochondrial NADH:ascorbate radical oxidoreductase in the extravesicular medium causes a decrease in intravesicular ascorbate radical in chromaffin granule ghosts but not in liposomes. These data provide direct experimental evidence for the hypothesis that the adrenal medullary mitochondrial NADH:ascorbate radical oxidoreductase could drive the re-reduction of ascorbate free radical generated inside the chromaffin granule by the turnover of dopamine beta-hydroxylase, without the ascorbate radical ever having to leave the granule.

Animals↗
PubMed1986

Biochemical investigation of human tumours in vivo with phosphorus-31 magnetic resonance spectroscopy.

The bioenergetic state of 15 human tumours was examined with phosphorus-31 magnetic resonance spectroscopy. A striking diversity in metabolic patterns was observed, and significant differences from normal tissue were seen in all cases. A common feature was an elevation of intracellular pH, which may be related to an increase in Na+/H+ exchange during cell activation. It is unlikely that the patterns observed directly correlate with malignancy, but characterisation of the energetic state of a given tumour in a given physiological environment may help in the design and evaluation of interventions for that specific case.

Adult↗
PubMed1986

An investigation of arterial insufficiency in rat hindlimb. A combined 31P-n.m.r. and bloodflow study.

A small animal model of arterial insufficiency is presented which involves unilateral femoral artery ligation and section. Invoked alterations in metabolism and perfusion of the affected muscle mass have been investigated 12 h, 4, 7 and 14 days post-ligation by 31P-n.m.r. and microsphere infusion, both at rest and during isometric muscle contraction at 1 Hz. At rest, the concentration of phosphocreatine was similar to the mean control value (36.0 +/- 1.0 mM) from 4 days post-ligation, but was significantly lower at 12 h (28.5 +/- 3.6 mM). Inorganic phosphate concentrations were significantly elevated for 7 days post-ligation. No significant differences were noted in intramuscular pH. Upon stimulation of the affected muscle mass, a time-dependent improvement in phosphocreatine utilization was observed such that 14 days post-ligation phosphocreatine utilization was not significantly different from mean control values. A similar amelioration was noted for the contraction-induced fall in intramuscular pH. At rest, no significant differences in bloodflow to the muscles of the ligated limb compared with the unaffected contralateral limb were observed. However, isometric contraction of the affected muscle mass resulted in a markedly reduced hyperaemic response 12 h post-ligation. Thereafter, a time-dependent improvement in tissue perfusion during stimulation was observed which paralleled the improvements in phosphocreatine utilization and intramuscular pH changes. The results presented are discussed with respect to the interrelationship between oxygen delivery, high energy phosphate utilization and force maintenance.

Animals↗
PubMed1986

An investigation of arterial insufficiency in rat hindlimb. An enzymic, mitochondrial and histological study.

A small animal model of arterial insufficiency has been used to investigate enzymic alterations in the gastrocnemius, plantaris and soleus muscles of the hypoperfused limb. At 7 days after induction of arterial insufficiency by unilateral femoral artery ligation, there were significant increases in the maximal activities of hexokinase, phosphorylase and 6-phosphofructokinase, whereas the activities of citrate synthase and 2-oxoglutarate dehydrogenase remained unchanged. Similar increases in hexokinase, phosphorylase and 6-phosphofructokinase were still apparent 8-10 weeks after unilateral artery ligation, although only hexokinase remained significantly higher than contralateral control values. No enhancement of oxidative enzyme activities was observed. The results are discussed in relation to the conflicting findings reported by other groups investigating enzymic adaptations in patients with arterial insufficiency.

Animals↗
PubMed1986

The metabolic state of the rat liver in vivo measured by 31P-NMR spectroscopy.

Previous 31P nuclear magnetic resonance (NMR) studies have measured the concentrations of phosphates, free Mg2+, pH and flux through enzyme-catalyzed reactions in a variety of tissues. A surgically-implanted coil has been developed to measure these parameters in the rat liver in vivo, and to assess the effect of external perturbations on the concentrations and physiological environment of phosphorus metabolities in the liver. The sensitive volume and optimal pulse were determined for the coil, which was insulated to exclude signal from surrounding tissues. The metabolic stability of the liver during acquisition of spectra was demonstrated by normal values for [Pi], [ATP], [lactate], and [pyruvate] in livers which were freeze-clamped immediately after completion of the NMR experiment. The stability was also confirmed by constant values for intracellular pH (7.2), free [Mg2+] (0.7 mM), and NMR detectable [Pi]/[ATP]. The sensitivity of the 31P-NMR spectrum of the liver in vivo to the physiological state of the animals was illustrated by comparing spectra from fed and 48 h fasted rats. The major qualitative differences were an increase in the pyridine nucleotide/adenine nucleotide ratio, and a small, but consistent shift in the frequency of the composite phosphomonoester peak. The spin-lattice relaxation time of each major phosphate resonance was measured in vivo using a modified homospoil saturation recovery pulse sequence; the T1 of ATP gamma-phosphate was 0.17 s. Selective saturation experiments did not detect magnetization transfer between the ATP gamma-phosphate and inorganic phosphate.

Adenine Nucleotides↗
PubMed1986

Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy.

The effects of 48 h fasting, administration of ethanol or 2,4-dinitrophenol, on the phosphorus-containing metabolites in liver in vivo have been determined utilizing 31P nuclear magnetic resonance spectroscopy. These measurements were combined with determinations of metabolite concentrations in livers which were freeze-clamped immediately after the NMR measurements were completed. Administration of sub-lethal amounts of dinitrophenol dramatically decreased ATP and increased Pi concentrations in liver in vivo as indicated by a 2.7-fold increase in the NMR-derived [Pi]/[ATP] ratio. Ethanol administration to fed animals increased the NMR-derived [Pi]/[ATP] ratio 27%; in contrast, the same amount of ethanol administered to fasted animals decreased the NMR-derived [Pi]/[ATP] ratio 30%. The NMR visible Pi and ADP represent about 50% and 15% of the total Pi and ADP, respectively. The phosphorylation potentials calculated from the NMR visible Pi and ADP were an order of magnitude higher than those obtained from metabolite concentrations in freeze-clamped tissue. There was no apparent correlation between the phosphorylation potentials derived from either the NMR spectral analyses or from metabolite concentrations and the hepatic [NAD+]/[NADH] ratio. The chemical shift of Pi indicated that ethanol administration elicited a decrease in pH of 0.1 unit in liver in vivo. Hepatic free [Mg2+] was increased 21% in fasted animals, but was unaffected by ethanol administration.

Acetoacetates↗
PubMed1986

Energetics of human muscle: exercise-induced ATP depletion.

The energetics of human muscle have been investigated in vivo during and after fatiguing aerobic, dynamic exercise. Changes in cytoplasmic pH and concentrations of phosphocreatine, ATP and Pi were followed using 31P nuclear magnetic resonance spectroscopy. ATP was significantly depleted in 6 out of 12 experiments and in these 6 experiments decreased to 55 +/- 5% of the pre-exercise concentration. Depleted muscle had a lower phosphocreatine concentration (17 +/- 5% of resting value) and lower pH (6.12 +/- 0.04) than fatigued muscle in which ATP loss was not observed (26 +/- 5% for phosphocreatine and 6.37 +/- 0.09 for pH). The free energy of hydrolysis of ATP was not significantly different in the two groups and was also similar in exhausted and nonexhausted muscle. Loss of ATP was associated with altered recovery of the muscle: [phosphocreatine], [Pi], and pH returned more slowly to their pre-exercise values and the initial rate of oxidative phosphorylation was diminished. The restitution of [ATP] to its pre-exercise value was much slower than that of the other metabolites.

Adenosine Diphosphate↗
PubMed1986

Are quenches dangerous?

There exists some uncertainty about the hazards which the quenching of a superconducting magnet would present to a subject undergoing an NMR examination. To investigate this problem, a 1.6-T whole-body magnet was quenched with an anesthetized pig lying in the bore. This paper reports our findings which, in the circumstances of this experiment, suggest that the risks are small.

Animals↗
PubMed1986

Myoglobin-dependent oxidative metabolism in the hypoxic rat heart.

The role of myoglobin in facilitating O2 diffusion for oxidative energy production was investigated at high (0.9 mM) and low (0.1 mM) O2 tensions in the Langendorff-perfused rat heart. 31P nuclear magnetic resonance was used to monitor the intracellular pH and concentrations of high energy phosphates. NaNO2 or phenylhydrazine was used to inactivate greater than 85% of intracellular myoglobin. During hypoxia, ATP and phosphocreatine were depleted significantly more rapidly in hearts with reduced concentrations of functional myoglobin than in control hearts. However, at 0.9 mM O2, myoglobin inactivation did not limit oxidative energy metabolism. It is concluded that facilitation of O2 diffusion by cardiac myoglobin plays a significant role in O2 delivery to the mitochondria at low O2 tensions.

Animals↗
PubMed1986

Acute renal failure in hemorrhagic hypotension: cellular energetics and renal function.

In male Wistar rats, renal adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH were measured by 31phosphorus nuclear magnetic resonance (31P NMR) and correlated with renal function before, during, and for one hour after a period of 30 to 40 minutes hemorrhagic hypotension. In animals which suffered no change in these metabolites during hypotension, retransfusion immediately restored normal renal function. When metabolite changes were observed during hypotension, they occurred suddenly with severe ATP depletion, Pi accumulation, and intracellular acidosis occurring almost concurrently. Metabolic changes of this magnitude were always associated with renal dysfunction in the post-hypotensive period, which occurred even when the period of biochemical change was only 10 to 15 minutes. The abnormalities in post-hypotensive renal function resemble the pattern of change seen in human acute tubular necrosis (ATN): depressed glomerular filtration rate (GFR), urine output varying from polyuria to oliguria, decreased urine to plasma inulin ratio, increased urinary sodium concentration, increased fractional excretion of sodium, and increased fractional excretion of potassium. It is postulated that changes in renal cellular energy status during hemorrhagic hypotension distinguish pre-renal failure from early or incipient ATN.

Acute Kidney Injury↗
PubMed1986

Muscle metabolism in patients with peripheral vascular disease investigated by 31P nuclear magnetic resonance spectroscopy.

Eleven men with claudication and ten control subjects had calf muscle metabolism studied at rest and during exercise and the subsequent recovery period by 31P nuclear magnetic resonance (n.m.r.) spectroscopy. The muscle of patients with severe claudication had a significantly greater depletion of phosphocreatine and fall in pH during exercise and a slower recovery of phosphocreatine and pH after exercise. The muscle of patients with both mild and severe disease had slower rates of ADP recovery after exercise than that of control subjects. Surgical correction of the associated arterial stenosis abolished claudication and led to correction of the metabolic abnormalities in two patients. Claudication pain was not related to intracellular pH or concentration of phosphorus-containing metabolites. Energy production via oxidative metabolism is impaired but glycolysis may be increased in the calf muscle of patients with intermittent claudication.

Adenosine Diphosphate↗
PubMed1986

A study of intracellular orthophosphate concentration in human muscle and erythrocytes by 31P nuclear magnetic resonance spectroscopy and selective chemical assay.

In order to study the relationship between extracellular and intracellular concentrations of orthophosphate (Pi), phosphorus nuclear magnetic resonance spectra were recorded, at rest, from the flexor digitorum superficialis muscle of hypophosphataemic patients with vitamin D-resistant rickets, and patients with Paget's disease of bone before and after they had been made hyperphosphataemic by treatment with the drug ethylidene-1-hydroxy-1,1-bisphosphonate. Changes in intramuscular P1 were estimated from the ratio of the areas of the Pi to adenosine 5'-triphosphate peaks. Even though the plasma Pi concentration in these patients spanned a fourfold range (0.5-2.0 mmol/l) the corresponding intramuscular Pi concentration increased by only 70%. A similar effect was observed in erythrocytes, from patients with these disorders, which were incubated in autologous plasma at 37 degrees C, under an atmosphere of O2 + CO2 (95:5, v/v). However, chloride ions, which are transported passively across the cell membrane, showed no change in distribution between cells and plasma, indicating that there was no general effect on passive anion distribution. When erythrocytes from normal subjects were incubated in autologous plasma (1.0 mmol of Pi/l) and in plasma supplemented with Pi (2.3 mmol of Pi/l), the Pi concentration in the cells, at steady state, increased only from 0.57 to 0.78 mmol/l cells, suggesting that the effect was not an artifact of disease or drug therapy. It is concluded that, in human skeletal myocytes and erythrocytes, the percentage change in the concentration of cytoplasmic Pi is lower than that in plasma. This implies that these cells can buffer or regulate cytoplasmic Pi when the extracellular concentration is disturbed.

Adult↗
PubMed1986

Biochemical mechanisms of acute contractile failure in the hypoxic rat heart.

The biochemical mechanism of acute contractile failure in the hypoxic rat heart was investigated using phosphorus nuclear magnetic resonance to measure intracellular acidosis and the concentrations of phosphocreatine, adenosine triphosphate (ATP), and inorganic phosphate while cardiac mechanical function was simultaneously monitored. The cytosolic free [ADP] was calculated from the creatine kinase equilibrium expression. Mechanical activity, phosphocreatine and ATP concentrations, and intracellular pH all decreased after the onset of hypoxic perfusion. Neither a reduction in ATP concentration nor limitation in its rate of production contributed to the early contractile failure. Calculations suggest only a modest (approximately 10%) difference in cytosolic free energies of ATP hydrolysis. Neither the time course nor the extent of depression of mechanical function correlated well with intracellular acidosis. In conjunction with other observations, these results were consistent with the view that the myocardial inotropic state may be directly responsive to the ambient PO2. The overall rate of ATP turnover was assessed from measurements of oxygen utilisation and lactate production in both normoxic and hypoxic hearts. Surprisingly, despite more than an 80% reduction in mechanical activity during hypoxia, no significant decrease in the rate of ATP utilisation was noted during hypoxia. This suggests that unidentified non-contractile processes may hydrolyse ATP at relatively higher rates during hypoxia.

Adenosine Triphosphate↗
PubMed1986

Influence of propranolol on acidosis and high energy phosphates in ischaemic myocardium of the rabbit.

Catecholamines stimulate adenosine triphosphate consumption and glycogenolysis in isolated hearts. In ischaemic myocardium a protective effect of beta adrenergic blockade may therefore arise from reduced adenosine triphosphate consumption or attenuation of acidosis. To characterise the biochemical mechanism of this protective effect phosphorus metabolite concentrations and intracellular pH were measured in an ischaemic region of the rabbit heart in vivo using 31P-nuclear magnetic resonance spectroscopy. After occlusion of the left anterior descending coronary artery there was a rapid decrease in intracellular phosphocreatine concentrations, and intracellular adenosine triphosphate concentrations decreased at a rate of approximately 0.5 mumol X g-1 dry weight X min-1. Intracellular pH decreased approximately linearly to a final pH of about 5.8. In the ischaemic myocardium of the animals treated with propranolol the intracellular concentrations of adenosine triphosphate were higher and those of phosphocreatine lower and the pH was the same compared with control after 30 minutes of ischaemia. Thus any protective effect of propranolol in vivo is not associated with attenuation of intracellular acidosis in this preparation.

Adenosine Triphosphate↗
PubMed1986

Assessment of human liver metabolism by phosphorus-31 magnetic resonance spectroscopy.

Phosphorus 31 magnetic resonance (MR) spectroscopy was used for the study of liver metabolism in vivo in seven healthy subjects. Subjects were examined in a 1.6 T whole-body magnet using surface coils for data acquisition. The region of the liver from which MR signals were collected was selected by magnetic-field profiling. The concentration ratios of adenosine triphosphate (ATP), inorganic phosphate (Pi) and sugar phosphates contained in liver cells could be reproducibly assessed. Cytosolic pH and the free magnesium concentration were determined to be 7.18 and 300 microM, respectively. During intravenous fructose tolerance tests the hepatic concentrations of sugar phosphates, ATP and Pi altered markedly. During the first 5 min following bolus injection of 250 mg fructose/kg body weight the concentration of sugar phosphates increased sevenfold whereas Pi and ATP decreased by three- to fourfold. Metabolism of sugar phosphates was complete within 20 min and could be followed by 31P MR with a time resolution of 5 min. Thus, 31P MR spectroscopy yields insight into liver metabolism which has not been accessible so far using conventional non-invasive methods. In conjunction with intravenous fructose loading, 31P MR spectroscopy may provide a means for the functional assessment of the liver.

Adenosine Triphosphate↗
PubMed1985

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.

Animals↗