oligomycins and Hypoxia

A groundbreaking publication by Sinclair and coworkers has illuminated the pseudo-hypoxic state in aging and its reversibility. Remarkably, these data also fit the mTOR-centered model of aging. Here we discuss that the mTOR pathway can cause cellular pseudo-hypoxic state, manifested by HIF-1 expression and lactate production under normoxia. We found that rapamycin decreased HIF-1 and lactate levels in proliferating and senescent cells in vitro. This reduction was independent from mitochondrial respiration: rapamycin decreased lactate production in normoxia, hypoxia, and in the presence of the OXPHOS inhibitor oligomycin. We suggest that pseudo-hypoxic state is not necessarily caused by mitochondrial dysfunction, but instead mitochondrial dysfunction may be secondary to mTOR-driven hyperfunctions. Clinical applications of rapamycin for reversing pseudo-hypoxic state and lactate acidosis are discussed.

Topics: Aging; Animals; Antibiotics, Antineoplastic; Cell Hypoxia; Cell Line; Cell Proliferation; Cellular Senescence; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lactic Acid; Mice; Mitochondria; Oligomycins; Oxidative Phosphorylation; Sirolimus; TOR Serine-Threonine Kinases

Stimulations of glucose transport produced by insulin action, contraction, or through a change in cell energy status are mediated by separate signaling pathways. These are the wortmannin-sensitive phosphatidylinositol 3-kinase pathway leading to the intermediate Akt and the wortmannin-insensitive AMP-activated protein kinase (AMPK) pathway. Electrical stimulation of cardiomyocytes produced a rapid, insulin-like, wortmannin-sensitive stimulation of glucose transport activity, but this occurred without extensive activation of Akt. Although AMPK phosphorylation was increased by contraction, this response was not wortmannin-inhibitable and consequently did not correlate with the wortmannin sensitivity of the transport stimulation. Oxidative metabolism stress due to hypoxia or treatment with oligomycin led to increased AMPK activity with a corresponding increase in glucose transport activity. We show here that these separate signaling pathways converge on GLUT4 trafficking at separate steps. The rate of exocytosis of GLUT4 was rapidly stimulated by insulin, but insulin treatment did not alter the endocytosis rate. Like insulin stimulation, electrical stimulation of contraction led to a stimulation of GLUT4 exocytosis without any marked change in endocytosis. By contrast, after oxidative metabolism stress, no stimulation of GLUT4 exocytosis occurred; instead, this treatment led to a reduction in GLUT4 endocytosis.

Topics: Androstadienes; Animals; Biological Transport; Biotinylation; Electrophysiology; Endocytosis; Exocytosis; Glucose; Glucose Transporter Type 4; Hypoxia; Insulin; Male; Monosaccharide Transport Proteins; Muscle Proteins; Myocytes, Cardiac; Oligomycins; Oxidative Stress; Oxygen; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Rats; Rats, Wistar; Receptors, Transferrin; Signal Transduction; Time Factors; Wortmannin

Adenosine is an endogenous nucleoside that signals through G-protein coupled receptors. Extracellular adenosine is required for receptor activation and two pathways have been identified for formation and cellular release of adenosine. The CLASSICAL pathway relies on intracellular formation of adenosine from adenine nucleotides and cellular efflux of adenosine via equilibrative nucleoside transporters (ENTs). The ALTERNATE pathway involves cellular release of adenine nucleotides, hydrolysis via ecto-5'-nucleotidases and extracellular formation of adenosine.. A rat model of cerebral ischemia and primary cultures of rat forebrain astrocytes and neurons were used.. Using a rat model of cerebral ischemia, the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) significantly increased post-ischemic forebrain adenosine levels and significantly decreased hippocampal neuron injury relative to saline-treatment. NBMPR-induced increases in adenosine receptor activation were not detected, suggesting that altering the intracellular:extracellular distribution of adenosine can affect ischemic outcome. Using primary cultures of rat forebrain astrocytes and neurons, adenosine release was evoked by ischemic-like conditions. Dipyridamole, an inhibitor of ENTs, was more effective at inhibiting adenosine release from neurons than from astrocytes. In contrast, alpha , beta-methylene ADP, an inhibitor of ecto-5'-nucleotidase, was effective at inhibiting adenosine release from astrocytes, but not from neurons. Thus, during ischemic-like conditions, neurons released adenosine via the CLASSICAL pathway, while astrocytes released adenosine via the ALTERNATE pathway.. These cell type differences in pathways for adenosine formation during ischemia may allow transport inhibitors to block simultaneously adenosine release from neurons and adenosine uptake into astrocytes. In principle, this could improve neuronal ATP levels without decreasing adenosine receptor activation.

Topics: Adenosine; Affinity Labels; Animals; Astrocytes; Brain Ischemia; Cells, Cultured; Deoxyglucose; Dipyridamole; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Glucose; Hypoxia; Inosine; Models, Biological; Neurons; Oligomycins; Phosphodiesterase Inhibitors; Prosencephalon; Purines; Rats; Thioinosine; Tritium

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN(-)) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca(2+), which was inhibited on removal of extracellular Ca(2+). Under current clamp conditions rotenone and CN(-) caused a rapid membrane depolarization and elevation of [Ca(2+)](i). Voltage clamping cells to -70 mV substantially attenuated this rise in [Ca(2+)](i). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K(+) currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K(+) current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN(-) or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

Topics: Animals; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carotid Body; Electron Transport; Enzyme Inhibitors; Hypoxia; Intracellular Membranes; Membrane Potentials; Methacrylates; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neurons; Oligomycins; Osmolar Concentration; Rats; Rats, Sprague-Dawley; Rotenone; Sodium Cyanide; Thiazoles; Uncoupling Agents

We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs.

Topics: Acetyl-CoA Carboxylase; Adenosine Triphosphate; AMP-Activated Protein Kinases; Animals; Blotting, Western; Cell-Free System; Dose-Response Relationship, Drug; Drosophila melanogaster; Genome; Humans; Hypoxia; Immunohistochemistry; Microscopy, Fluorescence; Multienzyme Complexes; Oligomycins; Peptides; Phosphorylation; Phylogeny; Precipitin Tests; Protein Serine-Threonine Kinases; RNA; RNA, Double-Stranded; Signal Transduction; Time Factors

To test the hypothesis that the voltage-insensitive background leak K+ channel is responsible for the oxygen-sensitive properties of glomus cells in the rat carotid body (CB) we used Ba2+, a non-specific inhibitor of K+ currents. In vitro changes in cytosolic calcium ([Ca2+]c) and chemosensory discharge were studied to measure the effect of Ba2+. In normal Tyrode buffer, Ba2+ (3 and 5 mM) significantly increased carotid sinus nerve (CSN) discharge over baseline firing rates under normoxia (PO2 approximately 120 Torr) from approximately 150 to approximately 600 imp/0.5 s. However, addition of 200 microM Cd2+ which completely blocked increase in CSN activity stimulated by hypoxia (PO2 approximately 30 Torr), hypercapnia (PCO2 approximately 60 Torr, PO2 approximately 120 Torr) and high CO (PCO approximately 550 Torr, PO2 approximately 120 Torr) did not significantly inhibit Ba2+-stimulated CSN discharge. The response to hypoxia is abolished with Ca2+-free tyrode buffer containing 10 mM EGTA. Yet, in the same buffer, Ba2+ increased CSN discharge from approximately 2 to approximately 180 imp/0.5 s. With 200 microM Cd2+ and 10 mM EGTA, Ba2+ still increased CSN discharge from approximately 2 to approximately 150 imp/0.5 s. Oligomycin (2 microg) abolished the hypoxic response. However, in the presence of oligomycin CSN response to Ba2+ was significant. Since Ba2+ increased neural discharge under conditions where hypoxia stimulated CSN discharge is completely abolished, we suggest that the effect of Ba2+ on CSN discharge may not have anything to do with the oxygen sensing mechanism in the CB.

Topics: Animals; Barium; Cadmium; Calcium Channels; Carotid Body; Chelating Agents; Egtazic Acid; Electron Transport; Ganglionic Blockers; Hexamethonium; Hypoxia; Mitochondria; Oligomycins; Potassium Channels; Rats; Stimulation, Chemical; Uncoupling Agents

The aim was to identify the major ATP source controlling the activity of sarcolemmal K(ATP) channels in ventricular cardiomyocytes.. K(ATP)-channel current (I(KATP)) was measured with the patch-clamp technique in either the whole-cell (glycogenolysis blocked by 10 mmol/l EGTA), cell-attached, or inside-out configuration.. In the absence of any substrate, I(KATP) (amplitude 31+/-4 nA; n=5) appeared spontaneously 520+/-160 s (n=6) after whole-cell access. This latency was shortened by exposure to anoxia (117+/-33 s, n=32) and even more by uncoupling (1-10 micromol/l FCCP; 25+/-3 s; n=13) while the amplitude was unchanged. During metabolic inhibition the latency was remarkably prolonged when the F1F0-ATPase was blocked by oligomycin, suggesting that under those conditions the F1F0-ATPase is the major ATP consumer. Glucose (5.5-20.0 mmol/l) in the bath solution did not affect the amplitude of I(KATP) but prolonged its latency compared to respective substrate-free conditions. However, I(KATP) was blocked immediately by mitochondrial substrates. FCCP also induced large I(KATP) in cell-attached measurements in either the absence or presence of glucose and oligomycin.. The activity of K(ATP) channels in cardiomyocytes of mice is controlled by a cytosolic [ATP] pool for which oxidative phosphorylation is the predominant ATP source.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cytosol; Egtazic Acid; Enzyme Inhibitors; Glucose; Glycogen; Heart Ventricles; Hypoxia; Mice; Mitochondria, Heart; Myocardium; Oligomycins; Oxidative Phosphorylation; Patch-Clamp Techniques; Potassium Channels; Proton-Translocating ATPases; Pyruvic Acid; Uncoupling Agents

Feeding beta-guanidinopropionic acid (GPA), a competitive inhibitor of creatine transport, decreases mortality and increases brain ATP stability in hypoxic mice. To study brain ATP metabolism in GPA-fed animals, respiratory rates were measured in grey matter and white matter slices as well as cerebral hemisphere mitochondria from GPA-fed mice and rats. Creatine kinase and adenylate kinase activities were measured in rat cerebral grey matter and white matter. Respiratory rates and oxidative phosphorylation were the same in GPA-fed mice and control mice. The adenylate kinase activity increased 50% and creatine kinase showed a small decrease in grey matter. In white matter, creatine kinase increased 50% while adenylate kinase decreased. Thus, GPA produces opposite adaptive changes in adenylate kinase and creatine kinase in grey matter and in white matter. These results suggest that the creatine kinase reaction in grey matter acts to regulate cellular ADP and ATP concentrations.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylate Kinase; Animals; Brain; Creatine Kinase; Drug Resistance; Female; Guanidines; Hypoxia; Mice; Mice, Inbred Strains; Mitochondria; Oligomycins; Oxygen Consumption; Potassium Chloride; Propionates; Rats; Rats, Sprague-Dawley; Respiration

It is known that oligomycin reduces the oxidative phosphorylation high-energy state or high-energy intermediates by inhibiting the formation of ATP without directly inhibiting electron transport, whereas metabolic uncouplers dissipate the high-energy state without net production of ATP. The metabolic hypothesis for O2 chemoreception in the carotid body (CB) predicts that 1) oligomycin should diminish O2 consumption and attenuate O2 chemoreception and 2) uncouplers should reverse the effect of oligomycin by increasing O2 consumption without restoring O2 chemoreception. These predictions were tested by simultaneously measuring CB chemosensory discharge from the sinus nerve and the rate of tissue O2 disappearance (dPO2/dt) during interruption of perfusate flow in perfused-superfused cat CB preparations (n = 9). O2 consumption was calculated from dPO2/dt. Flow-interruption responses were measured before and after oligomycin (1-microgram bolus) and subsequently after dinitrophenol (50 microM). Chemosensory responses to bolus injections of hypercapnic Tyrode solution, cyanide, or nicotine were also tested periodically. Oligomycin diminished dPO2/dt from -2.67 +/- 0.30 to -2.02 +/- 0.19 (SE) Torr/s (P < 0.004, paired t test) and reduced the maximal sensory response from 196 +/- 43 to 124 +/- 12 impulses/s (P < 0.002, paired t test) while augmenting the initial response to CO2. Dinitrophenol reversed the metabolic depressant effect of oligomycin but further suppressed the chemosensory response. These results confirm the above predictions and strengthen the metabolic hypothesis for O2 chemoreception in the CB.

Topics: 2,4-Dinitrophenol; Animals; Carotid Body; Cats; Cyanides; Dinitrophenols; Electric Stimulation; Female; Hypoxia; In Vitro Techniques; Male; Nicotine; Oligomycins; Oxygen Consumption; Uncoupling Agents

We have previously established that 21-day-old postnatal rat oligodendrocytes, maintained in monolayer culture and subjected to 6 h of hypoxia, show reversible inhibition of synthesis of alpha-hydroxy fatty acid and myelin basic protein but a dramatic induction of a 22-kDa protein, suggesting that this is a good model to study the mechanism of CNS demyelination caused by hypoxic injury. We now report that hypoxia also dramatically inhibits the basal protein kinase C-mediated phosphorylation of myelin basic protein and myelin 2',3'-cyclic nucleotide phosphohydrolase by 80%, but that the inhibition of phosphorylation can be reversed by addition of a protein kinase C activator, phorbol 12-myristate 13-acetate. The mechanism of action appears to involve the uncoupling of signal transduction at a site before phospholipase C, because hypoxia did not affect protein kinase C activity or its translocation to the membrane fraction. The most potent activator of phospholipase C (as measured by inositol phosphate release) was carbachol (muscarinic M1 receptor agonist), followed by L-phenylephrine (alpha 1-adrenergic receptor agonist) in normal oligodendrocytes. Excitatory amino acids and histamine were ineffective. Hypoxia for 6 h completely inhibited both muscarinic and alpha 1-adrenergic receptor-mediated inositol monophosphate release but did not affect phospholipase D-coupled phosphatidylethanol production in response to carbachol. We therefore conclude from this and earlier work that early, reversible changes in oligodendrocyte metabolism result not simply from ATP depletion, but may specifically target GTP binding protein-mediated processes.

Topics: Animals; Hypoxia; Inositol Phosphates; Myelin Basic Protein; Oligodendroglia; Oligomycins; Phospholipase D; Phosphorylation; Protein Kinase C; Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction

2-Oxoglutarate-supported rat liver mitochondria were loaded with moderate amounts of calcium and submitted to O2 deprivation and reoxygenation. In the presence of acetoacetate, anaerobic energy production maintained Ca2+ retention by mitochondria during the anoxia period unless the Pi concentration of the incubation solution was raised to 4-6 mM. Acetoacetate prompted Ca2+ release from O2-deprived mitochondria at elevated Pi levels, presumably due to occurrence of a permeability transition of the inner membrane. Providing 3-hydroxybutyrate and malate, together with acetoacetate, was found to delay the permeability transition until O2 was reintroduced, i.e., O2 triggered a paradoxical release of Ca2+ from mitochondria under these conditions. Whether initiated by O2 in the presence of Pi or by Pi under aerobic conditions, Ca2+ release was initially activated and subsequently inhibited or reversed in the presence of alpha-tocopherol (10-90 mumol.g protein-1). Similar effects were exerted by alpha-tocopherol during Pi-induced Ca2+ release from oligomycin-treated mitochondria supported by succinate (+ rotenone). In addition, the permeability transition was delayed by retinol (3-30 mumol.g protein-1) while beta-carotene, ubiquinone, and water-soluble antioxidants, including Trolox C, were ineffective. Other observations suggest that the Ca(2+)-releasing and/or -retaining effects of alpha-tocopherol and retinol may be independent from pro- and/or antioxidant activities. Effects resembling those of alpha-tocopherol were exerted by alpha-tocopherol succinate, which is devoid of antioxidant activity. Our data indicate that the permeability transition of Ca(2+)-loaded liver mitochondria may be triggered by O2, in the presence of ketone bodies, and affected by lipid-soluble antioxidants through mechanisms seemingly unrelated to free-radical generation or scavenging.

Topics: Acetoacetates; Anaerobiosis; Animals; Antioxidants; beta Carotene; Calcium; Carotenoids; Chromans; Hypoxia; Kinetics; Mitochondria, Liver; Oligomycins; Oxygen; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Ubiquinone; Vitamin A; Vitamin E

It has been hypothesized that, in oxygen-depleted myocardial cells, mitochondria are depolarized and the F1,F0-proton adenosinetriphosphatase (ATPase) catalyzes net ATP hydrolysis when the cells exhibit the signs of an aerobic-anaerobic metabolic transition, which are increased lactate formation and decline in high-energy phosphate reserves [W. Rouslin, C. W. Broge, and I. L. Grupp. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1759-H1766, 1990]. This hypothesis was tested by incubating isolated cardiomyocytes from the adult rat in substrate-free Tyrode solution (37 degrees C, pH 7.4) at a PO2 less than or equal to 0.1 Torr, i.e., 1,000-fold below the normal arterial level. At this deep hypoxia, the following results were found. 1) Lactate production was activated to maximal rates and high-energy phosphate contents decreased (aerobic-anaerobic metabolic transition). The inhibitor of the mitochondrial F1,F0-proton ATPase oligomycin, however, added upon establishment of hypoxia, did not slow down, as in the case of depolarized mitochondria, but moderately accelerated energy depletion. 2) Activation of mitochondrial ATP hydrolysis could be provoked in these hypoxic cells by addition of cyanide, antimycin A, and rotenone, i.e., specific inhibitors of certain sites of the respiratory chain. The enhancement of loss of ATP could be inhibited by oligomycin. The results demonstrate that states of deep hypoxia of the cardiomyocyte are possible in which it undergoes an aerobic-anaerobic metabolic transition, indicated by increased lactate formation and progressive loss of cellular energy reserves, and yet mitochondrial ATPase hydrolytic activity is not activated.

Topics: 2,4-Dinitrophenol; Aerobiosis; Anaerobiosis; Animals; Dinitrophenols; Electrons; Energy Metabolism; Hypoxia; Mitochondria, Heart; Myocardium; Oligomycins; Oxygen Consumption; Proton-Translocating ATPases

Neonatal rat oligodendrocyte (OLG) cultures exposed to 6 h of gradual, progressive hypoxia in a GasPak (BBL, Becton Dickinson) apparatus were not injured or metabolically impaired, but instead showed a specific inhibition of de novo synthesis (measured by [3H]palmitic acid labeling) of the major myelin component galactosylceramide (GalCer). De novo synthesis of the 2-hydroxy fatty acid GalCer (HFA-GalCer) species, which requires O2 for its synthesis, was most severely inhibited (by 65%), while non-hydroxy GalCer species (NFA-GalCer) were less affected. The synthesis of membrane glycerophospholipids and sphingomyelin was unaffected by hypoxia. Treatment of OLG with 12 nM oligomycin, an inhibitor of mitochondrial ATP synthesis, resulted in an inhibition (by 50-60%) of synthesis of all GalCer species. [3H]Palmitate labeling of NFA-ceramide, the ungalactosylated precursor of NFA-GalCer species, increased in both hypoxia and oligomycin treatments, suggesting that the conversion of newly synthesized ceramide to GalCer was blocked. Newly synthesized HFA-ceramide did not accumulate in OLG, but the small labeled HFA-ceramide pool present during hypoxia was not converted into HFA-GalCer. Pulse-chase studies indicated that NFA- and HFA-ceramides labeled during these treatments were available for galactosylation and could be converted into GalCer upon reoxygenation. [3H]Galactose labeling of NFA-GalCer species was enhanced 2-fold in hypoxia, in contrast to the inhibition seen with [3H]palmitic acid labeling. Thus, while de novo GalCer synthesis was blocked in hypoxia, galactosylation of pre-existing ceramide pools was actually enhanced. Our evidence suggests that hypoxia results in a reversible inhibition of transport of newly synthesized ceramide from its site of synthesis to its site of galactosylation, but causes an increase in galactosylation of subcellular pools of pre-existing ceramide.

Topics: Animals; Biological Transport; Cells, Cultured; Cerebrosides; Galactosylceramides; Galactosyltransferases; Hypoxia; In Vitro Techniques; Myelin Sheath; Oligodendroglia; Oligomycins; Rats; Time Factors

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Cells, Cultured; Cold Temperature; Electric Stimulation; Energy Metabolism; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Intracellular Membranes; Ischemia; Male; Mitochondria, Heart; Oligomycins; Ouabain; Proton-Translocating ATPases; Rats; Rats, Inbred Strains; Vanadium

Effect of WEB 1881 FU on hypoglycemia/hypoxia-induced brain damage in rats was evaluated and compared to findings obtained with idebenone. We used an in vitro model that facilitated the direct monitoring of dopamine release from striatal slices. The response to high K+ stimulation under perfusion of the slices with D-glucose-free Ringer solution (hypoglycemia) decreased at 40 min, and then practically disappeared. WEB 1881 FU at 10(-6) M or idebenone at 10(-6) M significantly protected against impairment of the striatal responses under the conditions of hypoglycemia. Hypoglycemic injury, evidenced by a remarkable neuron loss, necrosis and spongyosis was also ameliorated by these drugs. WEB 1881 FU at 10(-6) M had a protective action against the impairment of striatal responses evoked by NaCN (electron transport inhibitor at site 3) and oligomycin (inhibitor of mitochondrial ATP synthesis), but idebenone at 10(-6) M did not. In light of these observations, the possibility that WEB 1881 FU and idebenone exert neuroprotective actions against hypoglycemic/hypoxic brain injury by activating energy metabolism with different mechanisms from each other has to be considered.

Topics: Animals; Benzoquinones; Brain Diseases; Corpus Striatum; Cyanides; Dopamine; Hypoglycemia; Hypoxia; In Vitro Techniques; Male; Neurons; Oligomycins; Parasympatholytics; Potassium; Pyrrolidinones; Quinones; Rats; Rats, Inbred Strains; Ubiquinone

The effects of carotid chemoreceptor stimulation by intracarotid injections of sodium cyanide (NaCN, 30 micrograms), antimycin A (AMC, 10 micrograms) and dopamine (DA, 10 micrograms) on phrenic nerve activity were studied before and after oligomycin (200 micrograms) in the rabbit. The excitatory responses to NaCN and AMC were abolished after intracarotid administration of oligomycin, whereas the DA-induced phrenic depression was only slightly diminished. In addition, the effects of hypoxia on phrenic nerve activity were also studied before and after oligomycin (200 micrograms) in some animals with denervated one carotid sinus nerve. The hypoxia-induced phrenic excitation was greatly reduced after intracarotid administration of oligomycin. These results indicate that the chemoreflex phrenic responses induced by NaCN, AMC and hypoxia are probably related to the phosphate potential in the carotid body.

Topics: Animals; Antimycin A; Carotid Arteries; Chemoreceptor Cells; Dopamine; Dopamine Antagonists; Hypoxia; Oligomycins; Phrenic Nerve; Rabbits; Sodium Cyanide; Stimulation, Chemical

In brain, heart and kidney, cell work in the absence of oxygen has been thought to precipitate anoxic damage by increasing the rate of depletion of cellular energy stores. In the medullary thick ascending limb of isolated perfused rat kidneys, however, reduction of ATP synthesis by a variety of mitochondrial or metabolic inhibitors caused ATP depletion comparable to that produced by oxygen deprivation but did not reproduce the lesions of anoxia. In these cells, unrestrained mitochondrial activity may be an important source of anoxic injury.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Cyanides; Deoxyglucose; Energy Metabolism; Hypoxia; Kidney Medulla; Malonates; Mitochondria; Oligomycins; Oxygen Consumption; Rats; Rotenone

Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluted for their effects on PGE2 production and ATP levels. None of the inhibitors affected PGE2 synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE2 and ATP levels. This suggests that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather than an effect on oxidative phosphorylation. When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was omitted, PGE2 levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE2 levels, with a maximal effect at 10 mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis. These results indicate that oxygen (substrate) availability can limit inner medullary PGE2 production. In view of the low pO2 in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin production in this tissue.

Topics: Adenosine Triphosphate; Animals; Cyanides; Deoxyglucose; Dinitrophenols; Hypoxia; Kidney; Kidney Medulla; Oligomycins; Oxygen; Prostaglandin Antagonists; Prostaglandins E; Rats; Sodium Fluoride

1. During aerobic incubation in 5 mM glucose medium, 10(-5) M-DNP reduced the action potential duration and amplitude and the developed tension of guinea-pig ventricular muscle more rapidly and to a greater extent than anoxia.2. The DNP effect on electrical and mechanical activity was even more pronounced following prolonged anoxic incubation. Since the action potential duration and developed tension of anoxic ventricular muscle have previously been shown to be dependent on glycolytic ATP, and since the effects of DNP could not be duplicated with NaCN, it was concluded that DNP was exerting an effect in addition to its uncoupling of oxidative phosphorylation.3. Anoxic muscle was incubated with 10(-4) M-IAA or with 10(-4) M-IAA + 10(-4) M-DNP. The ATP content of IAA-treated muscle was significantly lower than control but in the presence of both IAA and DNP there was a further reduction in ATP and an increased lactate production.4. Sodium azide (10(-2) M), a potent inhibitor of mitochondrial ATPase, did not prevent the reduction of ATP in DNP-treated anoxic muscle.5. Ouabain (10(-7) M) partially prevented the rapid decline of action potential duration and developed tension of DNP-treated anoxic muscle. In addition, the glycoside partially blocked the DNP-induced break-down of ATP and stimulation of lactate production.6. Oligomycin (10 mug/ml.) partially prevented the reduction in action potential duration and developed tension of DNP-treated anoxic muscle.7. It was concluded that DNP induces an ;energy leak' by actively promoting the hydrolysis of an high energy glycolytic intermediate at least one step beyond the sites of ATPase inhibition by ouabain and oligomycin.

Topics: Action Potentials; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Azides; Cyanides; Dinitrophenols; Glucose; Guinea Pigs; Heart Ventricles; Hypoxia; In Vitro Techniques; Iodoacetates; Lactates; Muscle Contraction; Myocardium; Oligomycins; Ouabain; Oxidative Phosphorylation; Oxygen Consumption; Papillary Muscles; Ventricular Function

Topics: Adrenocorticotropic Hormone; Animals; Antimycin A; Cold Temperature; Dinitrophenols; Ethylmaleimide; Hypoxia; In Vitro Techniques; Iodoacetates; Male; Oligomycins; Pituitary Gland; Rats; Vasopressins

Topics: Action Potentials; Adenosine Triphosphate; Cells, Cultured; Cyanides; Electrophysiology; Heart; Hexoses; Hypoxia; Myocardium; Oligomycins; Ouabain; Potassium; Sodium; Time Factors

1. In cats under methoxyflurane, DNP and other metabolic inhibitors were tested on cortical neurones by iontophoresis from micropipettes.2. DNP, dinitro-o-cresol, iodoacetate, pentachlorophenol and oligomycin (uncouplers or inhibitors of oxidative phosphorylation), as well as moderate anoxia, blocked selectively and reversibly spontaneous firing and discharges evoked by ACh; responses evoked by glutamate were facilitated by moderate doses of DNP and blocked only by large amounts.3. Azide, cyanide, ouabain and strophanthidine had a mainly excitatory effect; the cardiac glycosides tended to depress more strongly responses to glutamate.4. Intracellular observations showed that DNP causes a sharp fall in electrical excitability, associated with a hyperpolarization and fall in membrane resistance.5. The hyperpolarizing action of DNP had a mean reversal level (E(DNP)) nearly 30 mV more negative than the resting potential; E(DNP) was identical with the mean reversal level for the depolarizing action of ACh, measured on the same cells.6. DNP had its usual hyperpolarizing effect on neurones whose IPSPs had been made positive by raising the internal [Cl]; the mean E(IPSP) was over 30 mV more positive than E(DNP).7. It is concluded that DNP lowers excitability by raising the membrane conductance to K(+) (g(K)) and that it blocks ACh responses selectively because ACh has a precisely opposite action on these neurones.8. In the Discussion, it is suggested that the rise in g(K) is mediated by an increase in internal free Ca(2+), caused by a slowing of mitochondrial activity, and that a similar mechanism may play a significant role in general anaesthesia.

Topics: Acetylcholine; Animals; Antimetabolites; Azides; Cardanolides; Cardiac Glycosides; Cats; Cerebral Cortex; Cresols; Cyanides; Dinitrophenols; Excitatory Amino Acid Antagonists; Hypoxia; Iodoacetates; Iontophoresis; Membrane Potentials; Neurons; Oligomycins; Ouabain

Topics: Acclimatization; Adenosine Triphosphatases; Alanine Transaminase; Altitude; Animals; Aspartate Aminotransferases; Dinitrophenols; Fructose-Bisphosphate Aldolase; Glutamates; Hypoxia; Magnesium; Malate Dehydrogenase; Male; Mitochondria, Liver; Oligomycins; Oxidative Phosphorylation; Rats; Succinates