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Biomedical subjects

I Hassinen

Publications and source records attributed to I Hassinen.

At least 19 recordsLinked to original sources
PubMed2001

Reversible ischemic inhibition of F(1)F(0)-ATPase in rat and human myocardium.

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.

Adenosine Triphosphate↗
PubMed2000

Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide.

Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.

Amino Acid Sequence↗
PubMed2000

The NADH oxidation domain of complex I: do bacterial and mitochondrial enzymes catalyze ferricyanide reduction similarly?

The hexammineruthenium (HAR) and ferricyanide reductase activities of Complex I (H+-translocating NADH:ubiquinone reductase) from Paracoccus denitrificans and bovine heart mitochondria were studied. The rates of HAR reduction are high, and its steady-state kinetics is similar in both P. denitrificans and bovine Complex I. The deamino-NADH:HAR reductase activity of Complex I from both sources is significantly higher than the respective activity in the presence of NADH. The HAR reductase activity of the bacterial and mitochondrial Complex I is similarly and strongly pH dependent. The pK(a) of this activity could not be determined, however, due to low stability of the enzymes at pH values above 8.0. In contrast to the high similarity between bovine and P. denitrificans Complex I as far as HAR reduction is concerned, the ferricyanide reductase activity of the bacterial enzyme is much lower than in mitochondria. Moreover, ferricyanide reduction in P. denitrificans, but not bovine mitochondria, is partially sensitive to dicyclohexylcarbodiimide (T. Yagi, Biochemistry 26 (1987) 2822-2828). On the other hand, the inhibition of ferricyanide reduction by high concentration of NADH, a typical phenomenon in bovine Complex I, is much weaker in the bacterial enzyme. The functional differences between the two enzymes might be linked to the properties of their binuclear Fe-S clusters.

Animals↗
PubMed1999

Generation of reactive oxygen species by human mesothelioma cells.

Malignant mesothelioma cells contain elevated levels of manganese superoxide dismutase (MnSOD) and are highly resistant to oxidants compared to non-malignant mesothelial cells. Since the level of cellular free radicals may be important for cell survival, we hypothesized that the increase of MnSOD in the mitochondria of mesothelioma cells may alter the free radical levels of these organelles. First, MnSOD activity was compared to the activities of two constitutive mitochondrial enzymes; MnSOD activity was 20 times higher in the mesothelioma cells than in the mesothelial cells, whereas the activities of citrate synthase and cytochrome c oxidase did not differ significantly in the two cell lines. This indicates that the activity of MnSOD per mitochondrion was increased in the mesothelioma cells. Superoxide production was assayed in the isolated mitochondria of these cells using lucigenin chemiluminescence. Mitochondrial superoxide levels were significantly lower (72%) in the mesothelioma cells compared to the mesothelial cells. Oxidant production in intact cells, assayed by fluorimetry using 2',7'-dichlorodihydrofluorescein as a fluorescent probe, did not differ significantly between these cells. We conclude that mitochondrial superoxide levels are lower in mesothelioma cells compared to nonmalignant mesothelial cells, and that this difference may be explained by higher MnSOD activity in the mitochondria of these cells. Oxidant production was not different in these cells, which may be due to the previously observed increase in H2O2-scavenging mechanisms of mesothelioma cells.

Amitrole↗
PubMed1992

Evaluation of reperfusion strategy for the globally ischaemic rat heart. Recovery of function and energy metabolism.

OBJECTIVE: The sequelae of myocardial ischaemia can in principle be alleviated by repeated reperfusion, but the accumulation of adenosine monophosphate (AMP) and the loss of interstitial adenosine may lead to adenylate depletion. Repeated oxidative stress could predispose the heart to reperfusion injury. The aim of this study was to investigate the effect of intermittent reperfusion on myocardial energetics and postischaemic function. METHODS: Isolated retrogradely perfused rat hearts were subjected to 20 min ischaemia, this being continuous in group I while the hearts in group II were reperfused for three 2 min periods at 5 min intervals. Function and energy metabolism were evaluated during the postischaemic reperfusion. RESULTS: Considerable efflux of adenosine compounds was seen during the final reperfusion, this being greater in group I than in group II, at 6.6(SEM 0.9) v 2.0(0.4) mumol.g-1 dry weight (p < 0.01). Tissue AMP, inorganic phosphate, and adenosine catabolites were higher in group I than in II after the ischaemic insult (p < 0.02), and ATP was higher in group II at the end of the final reperfusion (p < 0.05). All the hearts recovered; however, in group I the rate-pressure product was lower than in group II. CONCLUSIONS: Repetitive reperfusion, although short in duration, is beneficial in ischaemia in terms of lower adenylate loss and better postischaemic recovery. This should be taken into consideration when designing clinical reperfusion interventions.

Adenosine Diphosphate↗
PubMed1990

Mechanism of fatty acid effect on myocardial oxygen consumption. A phosphorus NMR study.

The calorigenic effect of fatty acids on the intact myocardium was investigated in isolated rat hearts perfused with a phosphate-free bicarbonate buffer. 31P-NMR spectra were accumulated in a Fourier transform high-field spectrometer during titration of the heart with varying concentrations of hexanoate. Oxygen consumption, coronary flow, left ventricular pressure development, heart rate and tissue surface fluorescence of nicotinamide-adenine nucleotides were monitored simultaneously. It was found that hexanoate within a range of 40 to 800 microM increased the reduction of NADH/NAD, increased oxygen consumption and increased the phosphocreatine/inorganic phosphate ratio in a concentration-dependent manner. The results suggest that the fatty acid-induced increase in oxygen consumption is not due to a primary effect on the cellular energy state resulting from a decrease in the P/O ratio or uncoupling of the mitochondria but due to an increase in the thermodynamic driving force.

Animals↗
PubMed1983

Increased prolyl 4-hydroxylase activity in the myocardium of endurance-trained mice.

Endurance training over 3, 10 or 20 days increased the activity of prolyl 4-hydroxylase (PH) in the left ventricle of mice. No increase was observed in the weight of the left ventricle, in galactosylhydroxylysyl glucosyltransferase activity or in hydroxyproline concentration. The increase in PH suggests that the synthesis of collagen increases during physiological adaptation of the heart to endurance exercise without changes in the ventricle weight or its total collagen content.

Animals↗
PubMed1983

Role of acetaldehyde and acetate in the development of ethanol-induced cardiac lipidosis, studied in isolated perfused rat hearts.

Isolated perfused rat hearts were used to study the effects of ethanol, acetaldehyde, and acetate on the cellular redox state and fatty acid metabolism in the myocardium. Ethanol had negligible effects on the cellular redox state but at high concentrations depressed the contractile activity and thereby secondarily the oxygen consumption. Acetaldehyde in concentrations below 50 microM had negligible effects on the redox state of the mitochondrial free NAD+/NADH couple, as studied by surface fluorometry of flavins and nicotinamide nucleotides. A reduction of NAD+ was observed with concentrations between 50 and 500 microM, while in the range of 0.5-1 mM the effect was biphasic, i.e., an initial reduction was followed by oxidation concomitantly with an increase in heart rate and peak systolic pressure. Acetate in millimolar concentrations caused in the coronary flow. A mitochondrial acetaldehyde dehydrogenase was revealed in the myocardium, having an apparent Km of 1.1 microM for acetaldehyde. Acetaldehyde in 50-microM concentration had no major effects on the uptake, oxidation, or lipid incorporation of oleate in the myocardium. Acetate in concentrations less than 2 mM did not affect the uptake of oleate into the myocardium, but did inhibit is oxidation and enhance its incorporation into tissue lipids in a dose-dependent manner. 2 mM acetate caused a 91% increase in oleate incorporation into tissue lipids over 30 min. The data can be interpreted as showing that acetaldehyde and acetate, the metabolites of ethanol, have metabolic effects on the myocardium, but only those of acetate are significant in concentrations encountered during ethanol oxidation in vivo. It is probable that acetate is involved in the development of ethanol-induced myocardial lipidosis, inhibiting the oxidation of fatty acids, and channelling them into the esterification pathway.

Acetaldehyde↗
PubMed1981

Plasma membrane phosphate transport and extracellular phosphate concentration in the regulation of cellular respiration in isolated perfused rat heart.

The role of extracellular Pi and transmembrane fluxes across the sarcolemma in the regulation of cellular respiration was studied in isolated Langendorff-perfused rat hearts. Extracellular phosphate did not significantly affect the oxygen consumption or cellular phosphorylation potential of the myocardium. K+-induced arrest was used to change the mechanical work load of the heart. Arresting the heart caused a rapid decrease in the unidirectional efflux of phosphate determined by in vitro prelabelling of the intracellular phosphate compounds with 32P and determining the specific radioactivity of the gamma-P of ATP, and the label appearance into the perfusion medium. At normal or elevated perfusate phosphate concentration there was a fairly slow net uptake of phosphate. The decrease in phosphate fluxes upon the K+-induced arrest was probably not due to a decrease in the transmembrane Na+ or K+ gradients because a further increase in the perfusate K+ concentration caused an increase in the K+ efflux to the levels observed in contracting hearts. The use of higher than normal concentrations of phosphate necessitated a lowering of the extracellular Ca2+ concentration, which caused a diminution of the oxygen consumption, accompanied by mitochondrial flavoprotein in the heart. This finding suggested that the extracellular Ca2+ concentration may be involved in the substrate level regulation of mitochondrial metabolism.

Animals↗
PubMed1978

Effect of chronic hypoxia on hepatic triacylglycerol concentration and mitochondrial fatty acid oxidizing capacity in liver and heart.

The effect of moderated hypoxia (50.5 kPa air) and severe hypoxia (40.8 kPa air) in vivo liver and heart triglyceride concentration and mitochondrial respiration rates was studied. Liver triglyceride concentrations increased in severe hypoxia from 7.3 mumol/g wet weight to 23.3 mumol/g wet weight over 7 days. After the period of seven days in severe hypoxia, the palmitate, octanoate and palmitoylcarnitine oxidation rates of mitochondrial suspensions were significantly reduced when the citric acid cycle was operative. No decrease in the fatty acid, fatty acyl-CoA or carnitine derivative oxidation was observed when only the beta oxidation system was studied. Mitochondria isolated from the heart or liver after seven days in severe hypoxia showed reduced respiratory control ratios, the decrease being from the normal 4.9 to 1.9 in the liver mitochondria using succinate as substrate. The reduction in respiratory control was mainly due to lowered State 3 respiration rates. Some reduction in the ratio was also observed in the fasting controls, from 5.8 to 3.4 with succinate. The respiratory control ratio could be partially normalized by the addition of albumin to the isolation medium for the liver mitochondria after severe hypoxia. Under these conditions, however, the State 4 respiration of the mitochondria from the hypoxic animals was higher than that for the controls.

Animals↗
PubMed1978

Metabolic adaptation to hypoxia. Redox state of the cellular free NAD pools, phosphorylation state of the adenylate system and the (Na+-K+)-stimulated ATP-ase in rat liver.

The effect of hypoxia (30 min 10% or 8% O2) on the phosphorylation state and redox state of the cytosol and mitochondria of rat liver were studied. Measurements were made both from normal animals and animals which had been exposed to the reduced partial pressure of oxygen (50.5 kPa or 40.8 kPa of air) for one or seven days. Cytostolic free NAD was reduced in the liver both in acute hypoxia and in hypoxia after one or seven days, i.e. the lactate/pyruvate and sn-glycerol-3-phosphate/dioxyacetonephosphate ratios increased markedly. A marked reduction in the mitochondrial free NAD pool occurred only in acute hypoxia and only a slight reduction was observed in animals kept at 40.8 kPa for one or seven days, as evaluated from the hepatic hydroxybutyrate/acetoacetate ratio. Liver ATP concentration decreased rapidly in acute hypoxia without any significant recovery during one or seven days at 40.8 kPa. The hepatic ATP/ADP X Pi ratio decreased significantly, with a simultaneous decrease in the total adenine nucleotide concentration. A tendency was observed for the ATP/ADP X Pi ratio to return to normal after seven days, i.e. the values in acute hypoxia were significantly smaller than those noted in hypoxia after seven days, demonstrating an adaptation of the energy metabolism during prolonged hypoxia. Hepatic (Na+-K+)-stimulated ATP-ase activity was not affected by hypoxia.

Acetates↗
PubMed1978

Hepatic triacylglycerol and fatty-acid biosynthesis during hypoxia in vivo.

Hepatic fatty acid biosynthesis and the activity of phosphatidate phosphatidate phosphohydrolase, the rate-limiting enzyme of triacylglycerol biosynthesis, were studied after hypoxic periods of 1 and 7 days under hypobaric conditions at 40.8 kPa. Phosphatidate phosphohydrolase activity increased 2-fold in the soluble fraction of the liver after one day at 40.8 kPa, but had returned to normal by 7 days. This was accompanied by a significant increase in liver triacylglycerol and sn-glycerol-3-phosphate. The phosphatidate phosphohydrolase activity increased continuously over 7 days in the pair-fed controls, probably due to the restriction on food. Measured as in vivo incorporation of 3H2O into lipids, the hepatic fatty acid synthesis rate increased somewhat in acute hypoxia, but returned to normal values during 7 days of hypoxia. Plasma free fatty acids increased markedly after 24 h in the fasting controls (90%) with a smaller increase in the hypoxic group (50%) due to peripheral lipolysis. Hepatic glycogen stores decreased in the hypoxic and fasting animals both after 1 and 7 days. It is concluded that hypoxia induces the accumulation of fat into the liver at least partly as a consequence of an increase in phosphatidate phosphohydrolase activity in the soluble fraction of the liver.

Animals↗
PubMed1977

Effect of hypoxia on mitochondrial mass and cytochrome concentrations in rat heart and liver during postnatal development.

Cytochrome concentrations of rat heart and liver mitochondria were measured postnatally from newborn animals to young adults. Mitochondrial cytochrome aa3 concentration increased shortly after birth in both tissues, this concentration being in a newborn animal 0.149 +/- 0.027 nmol/mg protein in liver and 0.333 +/- 0.082 nmol/mg protein in heart. The respective values from a two week old animal were 0.216 +/- 0.031 and 0.583 +/- 0.100. Postnatally cytochromes c+c1 and b increased markedly in the heart, but in the liver of newborn animals the cytochrome content was more close to the adult values. The amount of mitochondrial protein increased after birth, too. In a newborn animal the mitochondrial protein values were 39.7 +/- 3.6 mg/g wet weight in liver and 26.6 +/- 6.5 mg/g wet weight in heart. In adult animals the respective values were 71.5 +/- 4.8 and 80.7 +/- 13.1. The effect of postnatal hypoxia (10% O2, 24 h and 48 h) on liver and heart mitochondrial cytochrome concentration and protein values of newborn animals were investigated. During hypoxia the amount of mitochondrial protein remained about at the level of a newborn animal. The postnatal increase in the mitochondrial cytochrome concentration, which was smaller in the liver than in the heart, was also inhibited by hypoxia.

Age Factors↗