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PubMed1995

Oxygraphic evaluation of mitochondrial function in digitonin-permeabilized mononuclear cells and cultured skin fibroblasts of patients with chronic progressive external ophthalmoplegia.

For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal amount of digitonin for plasma membrane permeabilization was determined to be 5, 10, and 0.1 micrograms/10(6) cells, respectively. Using these concentrations the rate of respiration of permeabilized cells with the mitochondrial substrates succinate (+ rotenone) or glutamate + malate can be stimulated between two- and fourfold by ADP and inhibited by carboxyatractyloside. The maximal respiratory activities of well-characterized preparations of permeabilized mononuclear cells of five patients with chronic progressive external ophthalmoplegia were compared to healthy controls and a 30 to 50% decrease of the ADP-stimulated respiration rates with glutamate + malate and succinate + rotenone was detected. This is an indication for the presence of the mitochondrial defect in respiratory active blood cells. Additionally, for two of these patients the mitochondrial defects were proven to be detectable by the determination of maximal oxygen consumption rates of digitonin-permeabilized cultured skin fibroblasts. Therefore, the determination of maximal oxidation capacities of a well-defined cell population using strictly standardized conditions of digitonin permeabilization is judged as a useful and sensitive method for the elucidation of mitochondrial function in extramuscular tissue.

Adolescent↗
PubMed2002

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei.

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.

Animals↗
PubMed1997

Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes.

Ischemia/reperfusion causes oxidant injury in isolated cardiomyocytes without neutrophils or xanthine oxidase. Since these cells contain mitochondria, we hypothesized that the mitochondrial electron transport chain (ETC) causes this injury. To test this, we altered two conditions known to change within the mitochondria during ischemia/reperfusion-the extent of ETC redox-reduction and oxygen levels-and measured the resulting oxidant generation and injury. Specifically, we exposed cardiomyocytes for 1 h to the mitochondrial ETC inhibitors cyanide, antimycin, and rotenone and measured oxidant generation, using the intracellular fluorescent probe 2',7'-dichlorofluorescin (DCFH, sensitive to H2O2 and hydroxyl radicals). Inhibitors causing more extensive redox-reduction of the ETC (cyanide or antimycin) generated more oxidants than did partial ETC reduction with the inhibitor rotenone (10-fold v five-fold increases in DCFH oxidation). In addition, the DCFH oxidation caused by cyanide could be completely attenuated by the antioxidants 2-mercaptopropionylglycine (MPG) and 1,10 phenanthroline (PHEN). Finally, we tested the relevance of this oxidant generation on cell survival and contraction, with and without antioxidant interventions. Cell viability and contraction after 3-h recovery from cyanide exposure was significantly improved by either the addition of antioxidants, or by the "antioxidant" strategy of lowering O2 levels (i.e. from 150 to 3 tau) during the cyanide exposure (13.8% death with hypoxic cyanide v 48.6% cell death with normoxic cyanide). Collectively, these findings demonstrate that mitochondrial ETC carriers can cause significant oxidant injury, greatest when fully redox-reduced and exposed to oxygen, conditions known to occur in the transition from ischemia to reperfusion.

Animals↗
PubMed2000

Organotins induce apoptosis by disturbance of [Ca(2+)](i) and mitochondrial activity, causing oxidative stress and activation of caspases in rat thymocytes.

Di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) cause thymus atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of thymotoxicity by these chemicals. In vitro, a similar concentration-dependency has been observed. The purpose of the present research was to investigate the mechanisms underlying DNA fragmentation induced by these organotin compounds in freshly isolated rat thymocytes. As previously shown for TBTC, DBTC is also able to significantly increase intracellular Ca(2+) level ([Ca(2+)](i)). The rise in [Ca(2+)](i), already evident 5 min after treatment, was followed by a dose- and time-dependent generation of reactive oxygen species (ROS) at the mitochondrial level. Simultaneously, organotins induced the release of cytochrome c from the mitochondrial membrane into the cytosol. ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca(2+) chelator, or rotenone, an inhibitor of the electron entry from complex I to ubiquinone, indicating the important role of Ca(2+) and mitochondria during these early intracellular events. Furthermore, we demonstrated that rotenone prevents apoptosis induced by 3 microM DBTC or TBTC and, in addition, that both BAPTA and Z-DEVD FMK (mainly a caspase-3 inhibitor) decreased apoptosis by DBTC (already shown for TBTC). Taken together these data show the apoptotic pathway followed by organotin compounds starts with an increase of [Ca(2+)](i), then continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.

Animals↗
PubMed1983

Phencyclidine-dependent chemiluminescence of rat alveolar macrophages.

Phencyclidine (PCP) is a drug of abuse which is commonly taken by inhalation or snorting. By this pathway, it comes in direct contact with the alveolar macrophages (AM) which phagocytize inhaled particles. The activated AM produce reactive oxidative products and release photons with resulting chemiluminescence (CL) that can be quantified in the presence of luminol. The effect of PCP on this functional ability has been studied. PCP-treated rat-AM in vitro causes a two-to threefold increase in CL emission, after challenging with zymosan particles. This CL augmentation by PCP appears not to be associated with increased phagocytosis. Several metabolic inhibitors have been studied, and only rotenone has been found to inhibit CL production to a greater degree with PCP than in its absence. It may be assumed that the enhanced CL produced in the presence of PCP is due to activation of the drug by some reactive oxygen state or states that are affected by rotenone.

Animals↗
PubMed1976

Calcium uptake in preterminal central synapses: importance of mitochondria.

Energy dependent 45Ca2+ uptake in the synaptosomal preparation from guinea pig cortex has been investigated. 45Ca2+ uptake was stimulated by ATP, the absolute value of uptake being dependent on the extent of synaptosomal disruption caused by osmotic shock. A quantitative comparison of microsomal and mitochondrial ATP-dependent 45Ca2+ uptake showed that only mitochondria had a large enough capacity to account for the Ca uptake levels observed in the synaptosomal preparation. ATP-stimulated 45Ca2+ uptake in mitochondria, 'intact' and 'shocked' synaptosomes was inhibited by atractyloside, DNP, oligomycin and ruthenium red but unaffected by antimycin A and rotenone. This was interpreted as evidence that mitochondria were responsible for ATP-dependent synaptosomal Ca2+ uptake, the increase in uptake seen on osmotic lysis being due to the deocclusion of intraterminal mitochondria. Synaptosomal and mitochondrial 45Ca2+ uptake was also stimulated by the mitochondrial respiratory substrate glutamate; this uptake was sensitive to antimycin A, DNP, rotenone and ruthenium red but insensitive to atractyloside or oligomycin thus indicating it was of mitochondrial origin. No change in glutamate-dependent 45Ca2+ uptake was seen on osmotic lysis of the synaptosomes as the expected increase due to the release of occluded mitochondria was counterbalanced by the damaging effect of hypo-osmotic shock on the glutamate-stimulated 45Ca2+ uptake process.

Adenosine Triphosphate↗
PubMed1983

Calcium movements and intracellular calcium distribution in neoplastic GH3 cells.

Experiments were carried out to investigate the nature of the calcium homeostatic mechanisms in neoplastic GH3 rat pituitary cells. GH3 cells grown and maintained in Ham's F10 culture medium contained 35 nmoles calcium/mg cell protein. When stimulated by thyrotropin releasing hormone (TRH) or elevated K+ concentrations, only the latter caused cell calcium levels to rise although both resulted in hormone release. When exposed to EGTA, the GH3 cells lost calcium. When the temperature was lowered to 4 degrees C, the cells gained calcium and when rewarmed were able to extrude the previously accumulated calcium. The increased cell calcium following cold exposure could be blocked by prior treatment with rotenone. If rotenone was added subsequent to the cold exposure, it did not block the extrusion seen upon rewarming. In the absence of glucose in the medium, the GH3 cells took up more calcium upon exposure to 4 degrees C, and upon rewarming the cells could not return to their previous low levels. There are thus significant differences in calcium homeostasis between the neoplastic GH3 cells and their normal pituitary counterparts. When intracellular calcium was localized with the potassium pyroantimonate technique, there was calcium found in/on mitochondria, membrane bound vesicles and plasma membrane. Nuclear staining was sparse, and nucleolar staining was virtually absent. Upon stimulation with TRH, there was a decrease in mitochondrial calcium along with increases in both plasma membrane and nucleolar calcium levels. Since total calcium is unchanged, this indicates a significant calcium redistribution in response to TRH. The increased nucleolar calcium may reflect a calcium dependent increase in mRNA synthesis as has been reported. Since TRH presumably acts at a surface receptor, the increased plasma membrane calcium might be functionally related to receptor activation.

Animals↗
PubMed1991

Sequential damage in mitochondrial complexes by peroxidative stress.

The biochemical characteristics of the electron transfer chain are evaluated in purified non-synaptic ("free") mitochondria from the forebrain of 60-week-old rats weekly subjected to peroxidative stress (once, twice, or three times) by the electrophilic prooxidant 2-cyclohexene-1-one. The following parameters are evaluated: (a) content of respiratory components, namely ubiquinone, cytochrome b, cytochrome c1, cytochrome c; (b) specific activity of enzymes, namely citrate synthase, succinate dehydrogenase, rotenone-sensitive NADH: cytochrome c reductase, cytochrome oxidase; (c) concentration of reduced glutathione (GSH). Before the first peroxidative stress induction, the rats are administered for 8 weeks by intraperitoneal injection of vehicle, papaverine, delta-yohimbine, almitrine or hopanthenate. The rats are treated also during the week(s) before the second or third peroxidative stress. The cerebral peroxidative stress induces: (a) initially, a decrease in brain GSH concentration concomitant with a decrease in the mitochondrial activity of cytochrome oxidase of aa3-type (complex IV), without changes in ubiquinone and cytochrome b populations; (b) subsequently, an alteration in the transfer molecule cytochrome c and, finally, in rotenone-sensitive NADH-cytochrome c reductase (complex I) and succinate dehydrogenase (complex II). The selective sensitivity of the chain components to peroxidative stress is supported by the effects of the concomitant subchronic treatment with agents acting at different biochemical steps. In fact, almitrine sets limits to its effects at cytochrome c content and aa3-type cytochrome oxidase activity, while delta-yohimbine sets limits to its effects at the level of tricarboxylic acid cycle (citrate synthase) and/or of intermediary between tricarboxylic acid cycle and complex II (succinate dehydrogenase).(ABSTRACT TRUNCATED AT 250 WORDS)

Almitrine↗
PubMed1996

Mitochondrial involvement in schizophrenia and other functional psychoses.

Gene expression has been studied in post-mortem frontal cortex samples from patients who had suffered from schizophrenia and depressive illness. mRNA was extracted and characterised by translation and separation of the products by 2D gel electrophoresis. Post-mortem artefacts and the agonal experience did not affect the size distribution or amount of specific translation products. Four expression products were specifically reduced in samples from schizophrenics compared with normals. The expression of six products was altered in affective disorder, one in common with schizophrenia, two the same as in schizophrenia but increased. cDNA libraries were produced from the mRNA samples and 5 clones present at abnormal levels in schizophrenia identified by differential screening, isolated and sequenced. All the sequences encode mitochondrial transcripts; four encode mitochondrial rRNA and one the amino acid sequence of cytochrome oxidase sub-unit II. Increased cytochrome oxidase transcripts were found in a further set of mRNA extracts from schizophrenic patients including two who had not received neuroleptic medication. The effects of neuroleptic administration as exemplified by alpha-flupenthixol compared with the ineffective beta-flupenthixol were studied in experimental animals. It was found that 13 out of 28 clones whose levels were altered were mitochondrial in origin including rRNA, COX I & II and the NADH-Q reductase. Those encoding respiratory enzymes were at abnormally low levels as a result of alpha-flupenthixol administration. Measurements of the enzymic activity of cytochrome c oxidase in post-mortem frontal cortex of schizophrenics did not indicate any differences in overall activity but there was a decreased sensitivity to azide that was abolished by neuroleptics. Studies on NADH-cytochrome c reductase showed that schizophrenics whether medicated or not had a reduced rotenone sensitive activity that was compensated for by increased rotenone insensitive activity. We conclude that changes in mitochondrial gene expression are involved in schizophrenia and probably other functional psychoses.

Adult↗
PubMed2005

Role of mitochondria in the regulation of hypoxia-inducible factor-1alpha in the rat carotid body glomus cells.

Hypoxia-inducible factor-1alpha (HIF-1alpha) protein, a heterodimeric transcription factor that regulates transcriptional activation of several genes, is involved in adaptive responses to hypoxia. Earlier, we have reported that in carotid body (CB), the peripheral oxygen sensing organ, HIF-1alpha is up-regulated during hypoxia. One model proposes that an intact mitochondrial respiratory chain is necessary for this regulation of HIF-1alpha. To test this hypothesis in the CB glomus cells, we studied the effect of mitochondrial electron transport chain (ETC) inhibitors: rotenone (complex I; 1 microM), malonate (complex II; 0.5 M), antimycin A (complex III; 1 microg/ml), sodium azide (complex IV; 5 mM), and uncoupler of oxidative phosphorylation: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 mM) on HIF-1alpha expression during normoxia and hypoxia. Inhibitors and uncoupler of mitochondrial ETC abrogated hypoxia-induced HIF-1alpha expression in isolated glomus cells significantly (P < 0.001). Effect of rotenone during hypoxia was abolished by succinate (4 mM), a substrate for complex II. Further, HIF-1alpha expression was not altered by any of these mitochondrial inhibitors during normoxia. Taken together, these results strongly indicate that a functional mitochondrial ETC is required for the stabilization of HIF-1alpha, and further the connection between HIF-1alpha and mitochondria in CB oxygen sensing is reiterated.

Animals↗
PubMed1985

Oxygen dependence and subcellular partitioning of hepatic menadione-mediated oxygen uptake. Studies with isolated hepatocytes, mitochondria, and microsomes from rat liver in an oxystat system.

Using an oxystat system, menadione (2-methyl-1,4-naphthoquinone)-mediated oxygen uptake was investigated in isolated rat hepatocytes, in malate/glutamate-supplemented mitochondria, and in NADPH-reduced microsomes at steady-state oxygen partial pressures (pO2) between 0.1 to 100 mm Hg (0.2-150 microM O2). Menadione-mediated stimulation of oxygen uptake was half-maximal at pO2 of 0.5, 0.2, and 0.9 mm Hg, respectively. In hepatocytes and mitochondria half-maximal concentrations of menadione were 15 and 4 microM. However, in microsomes saturation with menadione was not reached at concentrations up to 300 microM. Antimycin A inhibited menadione-mediated oxygen uptake in hepatocytes and mitochondria by about three-fourths, while rotenone was without inhibitory effect; KCN inhibited practically completely. In mitochondria menadione-stimulated oxygen uptake was significantly inhibited by dicoumarol but further enhanced by the addition of ADP, even in the presence of rotenone. The results suggest that menadione-mediated hepatocellular oxygen uptake proceeds almost independently of pO2 in most regions of the liver lobule but that in areas of low pO2 such as the centrolobular regions limitation by oxygen may occur. They also demonstrate that in the intact hepatocyte menadione-mediated oxygen uptake predominantly (greater than 90%) results from electron transfer in the mitochondrial respiratory chain by menadione.

Animals↗
PubMed1987

Vanadate-dependent oxidation of pyridine nucleotides in rat liver microsomal membranes.

An enzymatic Na3VO4-dependent system for the oxidation of reduced pyridine nucleotides in purified rat liver microsomes was characterized. The system has a pH optimum of 6.5, and appears to be specific for vanadate, since activity in the presence of a related transition metal, molybdate, was not detected. Vanadate-dependent oxidation occurred with a concomitant consumption of O2 and, contrary to previous reports, preferred NADPH over NADH. At pH 6.5, the NADPH/NADH oxidase activity ratio was greater than 2:1. Sodium vanadate-dependent oxidation of NADH was inhibited by rotenone, antimycin A, NaN3, and NaCN. Conversely, Na3VO4-dependent NADPH oxidation was slightly affected by rotenone, but was insensitive to antimycin A, NaN3, NaCN, or quinacrine. Vanadate-dependent oxidation of either pyridine nucleotide was inhibited by the addition of either superoxide dismutase or catalase, indicating that both superoxide and hydrogen peroxide may be intermediates in the process. Linear sucrose gradient purification of the microsomes showed that the vanadate-dependent system for NADPH oxidation resides primarily in the endoplasmic reticulum. These studies indicate the existence of separate and distinct enzymatic systems for vanadate-stimulated oxidation of NADPH and NADH in mammalian microsomal membranes, and argue against an exclusive role of endogenous superoxide in the process.

Animals↗
PubMed1989

Effect of esters of succinic acid and other citric acid cycle intermediates on insulin release and inositol phosphate formation by pancreatic islets.

Esters of carboxylic acids are permeable to cells and once inside the cell are hydrolyzed to carboxylic acids. Methyl and ethyl esters of succinate and other citric acid cycle intermediates were tested to find out whether they are insulin secretagogues. Monomethyl succinate stimulated insulin release from pancreatic islets in a concentration-dependent manner with maximal release attained at a concentration of 10 mM. Dimethyl succinate (10 mM) was as effective as monomethyl succinate, but pyruvate methyl ester, monoethyl succinate, and dimethyl fumarate were ineffective as primary secretagogues. However, dimethyl fumarate potentiated both leucine- and leucine-plus-glutamine-induced insulin release. Glucose, leucine, leucine plus glutamine, and monomethyl succinate increased inositol tris-, bis- and monophosphate formation in pancreatic islets and antimycin A inhibited this formation. Since mitochondrial metabolism is probably essential for glucose-induced insulin release and the metabolism of succinate and leucine (without or with glutamine) involves mitochondrial respiration exclusively, these results might indicate that mitochondrial metabolism generates conditions or factors that are transmitted to the cytosol to increase inositol trisphosphate formation and thus calcium mobilization and insulin release. Since succinate is believed to enter metabolism at site II of the mitochondrial respiratory chain, it is interesting that rotenone, an inhibitor of NADH dehydrogenase and site I of the respiratory chain, was a potent inhibitor of monomethyl succinate-induced insulin released. Rotenone also inhibited leucine (plus or minus glutamine)-induced insulin release. These results indicate that beta cell metabolism of monomethyl succinate and leucine, like glucose, influences dehydrogenases that produce NADH.

Animals↗
PubMed1978

Proton electrochemical gradient and phosphate potential in mitochondria.

The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.

Adenosine Triphosphate↗
PubMed1983

Hormonal regulation of mitochondrial function. Description of a system capable of mimicking several effects of glucagon.

Isolated rat liver mitochondria were incubated at 0 degrees C in a medium consisting of 225 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM KH2PO4, 5 mM MgCl2 and 10 mM Tris-HCl, pH 7.4 (buffer 1) for 10 min, centrifuged and resuspended in 0.3 M sucrose. This treatment resulted in a stimulation of mitochondrial functions, mimicking several of the effects that follow glucagon treatment of the intact rat or isolated hepatocytes. Both phosphate and potassium are required for this effect; the addition of magnesium serves to enhance it. Mitochondrial respiration is essential for the development of the activated state as the stimulation is blocked by increasing concentrations of rotenone in the incubation. The intramitochondrial ATP/ADP ratio is increased, but when this increase was prevented by including low levels of rotenone or oligomycin in buffer 1, the stimulation of mitochondrial function was not diminished, thus demonstrating that an increased ATP/ADP ratio is not essential for activation. The rate of citrulline formation was unaffected by buffer 1 treatment unless glutamate was also included in the medium, indicating that control of this mitochondrial function differs from other functions studied.

Adenosine Diphosphate↗
PubMed1984

Control of oxidative phosphorylation in rat heart mitochondria. The role of the adenine nucleotide carrier.

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.

Adenine Nucleotides↗
PubMed1988

Local and metastatic tumor growth and membrane properties of LM fibroblasts in athymic (nude) mice.

LM fibroblasts grown in a chemically-defined, serum-free medium readily incorporated choline or one of three analogues of choline, namely N,N-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine into membrane phospholipids. The effect of these phospholipid manipulations in vitro on tumor growth and metastasis was examined in nude mice. Serum and choline-fed cells most frequently metastasized (74% and 68%, respectively), while frequency of lung metastasis was 46%, 42% and 17% in mice injected with cells fed with dimethylethanolamine, monomethylethanolamine, and ethanolamine, respectively. Metastases from cells cultured with serum, choline or dimethylethanolamine, but not from monomethylethanolamine or ethanolamine, were extensive and highly invasive. The specific activity of the (Na+ + K+)-ATPase but not of 5'-nucleotidase was significantly decreased in local tumor plasma membranes from choline analogue-fed cells as compared to tumor plasma membranes from choline-fed cells. When compared to the choline-fed tumor cells, the specific activities of three mitochondrial enzymes, namely NADH dependent, rotenone insensitive NADH-dependent, and rotenone sensitive NADH-dependent cytochrome-c reductase, were significantly increased in the choline analogue-supplemented cells. The arachidonic acid content of phosphatidylcholine in plasma membranes, microsomes, and mitochondria was significantly decreased in tumor membranes from choline analogue-fed cells as compared to tumor membranes from choline-fed cells. As compared to local tumor plasma membranes, the lung metastasis plasma membranes had elevated (Na+ + K+)-ATPase specific activity, phospholipid oleic and arachidonic acid content, and fluidity. In contrast, the 5'-nucleotidase specific activity, the content of cholesterol, phospholipid, and phosphatidylethanolamine were decreased in lung metastasis plasma membranes. In summary, membrane alterations of LM tumor cells in vitro (1) were not completely reversed in vivo, and (2) affected metastatic ability.

5'-Nucleotidase↗
PubMed1984

Reye's syndrome: salicylates and mitochondrial functions.

The effects of aspirin (acetylsalicylate, ASA) and related compounds in the presence of Ca2+ on the oxidative metabolism of isolated rat liver mitochondria were studied. Intact mitochondrial preparations preincubated with ASA + Ca2+ exhibited a transient stimulation of the state 4 respiratory rate with NAD+-linked substrates, followed by an inhibition which could not be released by the addition of ADP or uncoupler. Maximum respiratory rates were achieved by subsequent addition of NAD+ or succinate. The Ca2+-transport inhibitors ruthenium red and ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented these effects. Five brands of commercial aspirin were tested and were as effective as purified ASA. Tylenol (acetaminophen) could reproduce these effects only at much higher (greater than or equal to 10-fold) concentrations. Other salicyl derivatives showed results qualitatively similar to ASA, with potencies in the order: acid much much greater than ASA much greater than alcohol greater than or equal to catechol greater than amide, salicylate being approximately 10-fold more potent than ASA. The magnitude of the effect seen depended on the Ca2+ (endogenous + exogenous) and salicylate concentrations/mg mitochondrial protein, and on the length of the preincubation. Added inorganic phosphate was also required. That salicylate + Ca2+ induces an increase in the permeability of the mitochondrial inner membrane was demonstrated by the observation that 90% of the intramitochondrial NAD(P)+ was released into the surrounding medium upon preincubation of intact mitochondria with these agents. Salicylate + Ca2+ had virtually no effect on respiration with succinate (+ rotenone) as substrate at salicylate concentrations which markedly affected NAD+-linked substrate oxidation. The presence of rotenone in the preincubation mixture prevented the damaging effects of salicylate + Ca2+ on the mitochondrial membrane, suggesting that the redox state of intramitochondrial pyridine nucleotides can modulate these effects. The results reported here are similar to those reported previously by our laboratory for the effects of Reye's plasma and allantoin + Ca2+, and indicate that, like these agents, salicylate and salicyl compounds can potentiate the Ca2+-induced damage to the mitochondrial inner membrane and may be another factor responsible for Reye's syndrome.

Animals↗