Biomedical subjects
Publications and source records attributed to T Ramasarma.
Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.
On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58 kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50 kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58 kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50 kDa protein) to the membrane, present in the 58 kDa-protein appears to be involved in mediating the inhibition by silatrane.
The oxidation of NADH and accompanying reduction of oxygen to H2O2 stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H2O2, all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H2O2-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.
Polyvanadate solutions obtained by extracting vanadium pentoxide with dilute alkali over a period of several hours contained increasing amounts of decavanadate as characterized by NMR and ir spectra. Those solutions having a metavanadate:decavanadate ratio in the range of 1-5 showed maximum stimulation of NADH oxidation by rat liver plasma membranes. Reduction of decavanadate, but not metavanadate, was obtained only in the presence of the plasma membrane enzyme system. High simulation of activity of NADH oxidation was obtained with a mixture of the two forms of vanadate and this further increased on lowering the pH. Addition of increasing concentrations of decavanadate to metavanadate and vice versa increased the stimulatory activity, reaching a maximum when the metavanadate:decavanadate ratio was in the range of 1-5. Increased stimulatory activity can also be obtained by reaching these ratios by conversion of decavanadate to metavanadate by alkaline phosphate degradation, and of metavanadate to decavanadate by acidification. These studies show for the first time that both deca and meta forms of vanadate present in polyvanadate solutions are needed for maximum activity of NADH oxidation.
Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.
The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.
The oxidative activity of mitochondria freshly isolated from brown adipose tissue of rats was stimulated two-fold on the addition of small concentrations of exogenous cytochrome c to the reaction medium. Loss of membrane-bound cytochrome c did not occur during isolation of mitochondria. Estimation of the high-affinity binding sites on the organelle membrane indicated that less than a third of these sites remained saturated with cytochrome c. The pigment is thus shown to be a functionally limiting electron transport component in brown adipose tissue.
A concentration dependent inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase was found on preincubation of microsomal preparations with diallyl disulfide, a component of garlic oil. This inhibited state was only partially reversed even with high concentrations of DTT. Glutathione, a naturally occurring reducing thiol agent, was ineffective. The substrate, HMG CoA, but not NADPH, was able to give partial protection for the DTT-dependent, but not glutathione-dependent activity. The garlic-derived diallyl disulfide is the most effective among the sulfides tested for inhibition of HMG CoA reductase. Formation of protein internal disulfides, inaccessible for reduction by thiol agents, but not of protein dimer, is likely to be the cause of this inactivation.
Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 alpha-hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation. A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbent assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.
Perfusion of rat livers with polyvanadate, but not metavanadate, was found to increase in plasma membrane and decrease in cytosol protein kinase C activity, similar to that obtained with phenylephrine, an alpha-adrenergic agonist. The effect was prevented by phenoxybenzamine, but not by propranolol implicating alpha-adrenergic receptor activation. Comparison of crystal structures of decavanadate and nonadrenaline revealed the occurrence of a structural feature of O-O-O(N) with distances of 5.5 A and 2.9 A.
Exposure of rats to the cold (4-5 degrees C) caused large (2-3-fold) increases in the mass of interscapular brown adipose tissue (BAT), its mitochondrial content and the basal metabolic rate of the animals. The rate of substrate oxidation by BAT mitochondria also increased about 3-fold. When cold-acclimated animals were exposed to heat (37 degrees C), the BMR decreased by half in 3 h, the earliest time interval tested. Mitochondrial substrate oxidation, as well as substrate-dependent H2O2 generation, showed a proportionate decrease in rates. In these mitochondria, activities of cytochrome c reductases, but not dehydrogenases with NADH, alpha-glycerophosphate and succinate as substrates, also showed a significant decrease. The concentration of cytochromes aa3 and b, but not cytochrome c, also decreased in BAT mitochondria from 12-h heat-exposed animals, while the change in concentration of cytochrome b alone was found as early as 3 h of heat exposure. These results identify the change in cytochromes as a mechanism of regulation of oxidative activities in BAT mitochondria under conditions of acute heat stress.
Lipid peroxidation measured both by the formation of malondialdehyde and by oxygen uptake in presence of NADPH, Fe2+ and ADP in hepatic microsomes increased on cold exposure and decreased on heat exposure of rats. Malondialdehyde content of isolated microsomes also showed similar changes. Treatment of animals with noradrenaline or a alpha-adrenergic agonist, phenylephrine, increased lipid peroxidation which was prevented by simultaneous treatment with cycloheximide, a protein synthesis inhibitor. Cold- and noradrenaline-induced increases were not found in animals pretreated with alpha-adrenergic receptor antagonist, phenoxybenzamine, but not with propranolol, a beta-adrenergic blocking agent. The concentration of the microsomal cytochromes P-450 and b5 remained unaffected under these conditions but the activity of NADPH-cytochrome c reductase showed parallel changes. These observations suggest a role for lipid peroxidation in cellular thermogenesis in liver tissue.
The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.
H2O2, in addition to producing highly reactive molecules through hydroxyl radicals or peroxidase action, can exert a number of direct effects on cells, organelles and enzymes. The stimulations include glucose transport, glucose incorporation into glycogen, HMP shunt pathway, lipid synthesis, release of calcium from mitochondria and of arachidonate from phospholipids, poly ADP ribosylation, and insulin receptor tyrosine kinase and pyruvate dehydrogenase activities. The inactivations include glycolysis, lipolysis, reacylation of lysophospholipids, ATP synthesis, superoxide dismutase and protein kinase C. Damages to DNA and proteoglycan and general cytotoxicity possibly through oxygen radicals were also observed. A whole new range of effects will be opened by the finding that H2O2 can act as a signal transducer in oxidative stress by oxidizing a dithiol protein to disulphide form which then activates transcription of the stress inducible genes. Many of these direct effects seem to be obtained by dithiol-disulphide modification of proteins and their active sites, as part of adaptive responses in oxidative stress.
Acclimation of rats to cold caused 45% increase in the concentration of triidothyronine (T3) and 35% increase in the concentration of thyroxine (T4) in serum. Exposure of cold-acclimated rats to heat (12 hr, 37 degrees C) failed to decrease the concentrations of thyroid hormones in circulation. The concentration of T3 in brown adipose tissue (BAT) increased almost 10-fold on cold acclimation. Iodothyronine deiodinase activity also registered 3-fold increase. Exposure of cold-acclimated animals to heat caused decrease in the concentration of T3 in BAT without appreciably affecting T4 concentration. In liver tissue, the changes in hormone concentrations were quite small compared to those in BAT. On thyroidectomy or when fed with propyl thiouracil, rats could not survive exposure to the cold. The concentration of insulin in circulation showed small increase, while that in the tissues showed significant decrease on acclimation of rats to the cold. The concentration of the hormone in BAT registered significant increase on exposure of cold-acclimated animals to heat (12 hr, 37 degrees C). The increase in liver was marginal. The temperature-dependent response of T3 indicates an important role for this hormone in rapid physiological response in BAT.
Exposure of cold-acclimatized rats to heat (37 degrees C) for a short period decreased brown adipose tissue (BAT) mitochondrial substrate-dependent oxygen uptake and H2O2 generation. Both the concentration and substrate-dependent rate of cytochrome b reduction decreased as early as 3 h of heat exposure. These results identify cytochrome b as the locus of regulation of electron transport in BAT mitochondria under conditions of heat stress.
Mitochondria isolated from the livers of rats administered with sodium meta-, ortho-, or polyvanadate, but not vanadyl sulphate, exhibited enhanced Ca2+ -stimulated respiration and uptake of calcium. These effects were shown also by mitochondria isolated from livers perfused with polyvanadate. The concentration of acid-soluble calcium decreased significantly in the mitochondrial fraction on vanadate treatment, while that in the cytosol showed a corresponding increase. Phenoxybenzamine, an antagonist to alpha-adrenergic receptors, effectively inhibited vanadate-induced Ca2+ mobilization, but surgical sympathectomy was without effect. This is the first demonstration of vanadate mimicking alpha-adrenergic agonists in vivo.